Compositions and methods for deploying a transgenic refuge as a seed blend

ABSTRACT

Methods and compositions for deploying refuge seeds together with transgenic crop seeds are provided. The refuge seeds can be non-transgenic seeds of a similar variety to that of the transgenic crop seeds, or the refuge seeds can be a transgenic variety.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/780,151, filed Feb. 17, 2004, the entire disclosure of which isincorporated herein by reference in its entirety; which application is acontinuation-in-part of U.S. patent application Ser. No. 10/394,929filed Mar. 19, 2003, now abandoned which is a continuation of U.S.patent application Ser. No. 09/972,012, now U.S. Pat. No. 6,551,962filed Oct. 5, 2001, which claims priority to U.S. ProvisionalApplications Ser. Nos. 60/238,406 and 60/238,405, both filed Oct. 6,2000.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to transgenic crops and refugiaassociated with the transgenic crops, and more generally to the controlof pests that cause damage to crop plants, and in particular to peststhat cause damage by their feeding activities directed to roots, shoots,stems, flower parts, fruit and vegetable product parts, and leaf parts.The present invention is directed to methods of use of and compositionscomprising a first transgenic crop plant seed comprising one or moretransgenes which express one or more transgenic proteins in a seedmixture or seed blend with one or more refuge seeds, the refuge seedsbeing selected from non-transgenic seeds or from a second transgenicplant seed that acts as a seed that grows into a plant that exhibits thetraits of a refuge plant, but which contains one or more transgene(s)that is(are) different from that(those) in the first crop plant seed.Treatment of the first transgenic seed, the refuge seed, or both withone or more chemical herbicide agents, pesticide agents, peptides, ornucleic acids can be accomplished prior to planting of the seed blend toachieve the desired effect.

(2) Description of the Related Art

Insects, nematodes, and related arthropods annually destroy an estimated15% of agricultural crops in the United States and even more than thatin developing countries. In addition, competition with weeds andparasitic and saprophytic plants account for even more potential yieldlosses.

Some of this damage occurs in the soil when plant pathogens, insects andother such soil borne pests attack the seed after planting. In theproduction of corn, for example, much of the rest of the damage iscaused by rootworms—insect pests that feed upon or otherwise damage theplant roots; and by cutworms, European corn borers, other pests thatfeed upon or damage the above ground parts of the plant, and non-cropplants such as weeds and parasitic and saprophytic plants that candeprive the crop plant of valuable moisture and soil derived nutritionalpotential. General descriptions of the type and mechanisms of attack ofpests on agricultural crops are provided by, for example, Metcalf, inDestructive and Useful Insects, (1962); and Agrios, in Plant Pathology,3rd Ed., Academic Press (1988).

Corn is the most important grain crop in the Midwestern United States.Among the most serious insect pests of corn in this region are thelarval forms of three species of Diabrotica beetles. These include theWestern corn rootworm, Diabrotica vergifera vergifera LeConte, theNorthern corn rootworm, Diabrotica berberi Smith and Diabrotica berberiLawrence, and the Southern corn rootworm, Diabrotica undecimpunctatahowardi Barber. In fact, more chemical insecticide is used for thecontrol of corn rootworm than for any other pest of corn, and the totalacreage treated with chemical insecticides is greater than for any otherpest in the United States.

Corn rootworms (CRW) overwinter in the egg stage in fields where cornwas grown the previous season. The eggs hatch from late May throughJune. If a corn crop is not followed by another corn crop in thesubsequent year, the larvae will die. Accordingly, the impact of cornrootworm is felt most directly in areas where corn is systematicallyfollowed by corn, as is typical in many areas of the Midwestern UnitedStates.

After hatching, the larvae pass through three larval stages or instars,during which they feed on the corn root system. About three weeks isrequired for completion of the larval stage. Damage to the corn rootsystem caused by the feeding of larvae is the major cause of harvestlosses in corn due to corn rootworm. Corn plants that fall over andlodge in the field after weakening or destruction of a major part of theroot system are the cause of a major portion of this loss, since thislodged corn cannot be harvested by conventional mechanized machinery andis left in the field.

Following completion of larval development, the larvae transform intoimmobile pupae, and thence into the adult beetles that emerge from thesoil throughout the summer, with the period of emergence depending uponthe growing location. After emergence, the adult beetles feed for abouttwo weeks before the females start laying eggs. Initially, the adultsfeed predominantly in the same field from which they emerged, but laterwill migrate to other fields. Peak adult activity normally occurs in theU.S. Corn Belt during late July or early August in fields planted tocontinuous corn, but activity may peak later in first year or latematuring cornfields. Rootworm beetles begin depositing eggs incornfields approximately two weeks after they emerge. (For moreinformation, see, e.g., Corn Rootworms, Field Crops Pest ManagementCircular #16, Ohio Pest Management & Survey Program, The Ohio StateUniversity, Extension Division, Columbus, Ohio; available online at theOhio State web site ag.ohio-state.edu/˜ohioline/icm-fact/fc-16.html; andMcGahen et al., Corn Insect Control: Corn Rootworm, PENpages number08801502, Factsheet available from Pennsylvania State University, StateCollege, PA, 1989).

There is evidence of the emergence of a new race of Corn rootworm whichovipositions its eggs for overwinter onto adjacent soybean plants. Themost common practice in the mid-western united states has been forfields to be rotated annually with corn, followed the next year withsoybeans, in order to manage the development of an epidemic of cornrootworm pressure on fields of corn. While this strategy overall hasbeen successful in reducing the corn rootworm feeding pressure on cornin many areas, the evolutionary emergence of this new race of cornrootworm creates a problem which was not anticipated and which could nothave been easily foreseen. This new race, which preferentially depositsits eggs onto soybean fields, provides an unintended feeding pressure onthe next years' intended corn crop in the field in which soybeans weregrown the previous year, and the subsequent requirement for insecticidalcontrol measures which adds unintended cost to the farmer in the form ofadditional labor for spraying and additional costs of goods, furtherreducing the return to the farmer on his/her investment in the crop andharvest.

One means for combating the corn rootworm pressures in the US, inparticular in view of the introduction of recombinant crops containinggenes which express proteins which are insecticidal to a selected fewintended crop pest insect species, has been the regulatory agencies'requirement that farmers plant a non-recombinant refuge crop whichprovides a means for producing a steady and consistent population ofadult insects which have never been exposed to the recombinant pesticidepressures and so have not had the opportunity to develop resistance as aresult of the pesticide pressure when feeding on the recombinant plants.This is particularly true for the corn rootworm larvae as it is highlylimited in its ability to move through the soil any great distance fromthe roots which are more or less adjacent to its local larvalenvironment within the soil. In theory, the adult insects which emergefrom the refuge environment will disperse and breed with any insectswhich emerge from the recombinant fields, and if any of the insectswhich emerge from the recombinant fields have developed a level ofresistance to the recombinant insecticidal proteins, the availability ofthat trait in the subsequent generations will be diluted, reducing ordelaying the onset of the emergence of a race which will be totallyresistant to the recombinant insecticidal corn plant.

The western corn rootworm, D. virigifera virigifera, is a widelydistributed pest of corn in North America, and in many instance,chemical insecticides are indiscriminately used to keep the numbers ofrootworms below economically damaging levels. In order to assist in thereduction of chemical insecticides used in treatments to control therootworm populations in crop fields, transgenic lines of corn have beendeveloped which produce a one of a number of amino acid sequencevariants of an insecticidal protein produced naturally in the bacteriumBacillus thuringiensis. This protein, generally referred to as Cry3Bb,has recently been modified by English et al. in U.S. Pat. No. 6,023,013and related patents and applications, to contain one or more amino acidsequence variations which, when tested in insect bioassay against thecorn rootworm, demonstrates a from about seven (7) to about ten (10)increase in insecticidal activity when compared to the wild type aminoacid sequence. In particular, the enhanced expression of a gene encodingthis particular protein in root tissue in corn provides for improvedcorn rootworm control without the requirement for additional costs ofgoods by the farmer. In effect, a farmer planting corn rootwormprotected corn seeds would not have the costs of labor and of chemicalapplications in treating fields of corn crops to protect the fields fromcorn rootworm infestation.

As indicated above, one concern is that a race of rootworm will emergewhich has developed resistance to the Cry3Bb protein produced in thecorn plants.

One strategy for combating the development of resistance is to select arecombinant corn event which expresses high levels of the insecticidalprotein such that one or a few bites of a corn root would cause at leasttotal cessation of feeding and subsequent death of the corn rootworm.

Another strategy would be to combine a second corn rootworm specificinsecticidal protein in the form of a recombinant event in the sameplant, for example a recombinant acyl lipid hydrolase or insecticidalvariant thereof (WO 01/49834), a CryEt70, a Cry22, a CryEt33 and CryET34binary toxin complex, a PS 149B1 binary toxin complex, or a CryET80 andCryET76 binary toxin complex, along with a variant Cry3Bb insecticidalprotein. Preferably the second toxin or toxin complex would have adifferent mode of action from the Cry3Bb variant, and preferably, ifreceptors were involved in the toxicity of the insect to the recombinantprotein, the receptors for each of the two or more insecticidal proteinsin the same plant would be different so that if a change of function ofa receptor or a loss of function of a receptor developed as the cause ofresistance to the particular insecticidal protein, then it should notand likely would not affect the insecticidal activity of the remainingtoxin which would be shown to bind to a receptor different from thereceptor causing the loss of function of one of the two insecticidalproteins cloned into a plant.

Still another strategy would combine a chemical pesticide with apesticidal protein expressed in a transgenic plant. This couldconceivably take the form of a chemical seed treatment of a recombinantseed which would allow for the dispersal into a zone around the root ofa pesticidally controlling amount of a chemical pesticide which wouldprotect root tissues from target pest infestation so long as thechemical persisted or the root tissue remained within the zone ofpesticide dispersed into the soil. So long as root tissue was within thezone of chemical pesticide protection, a target pest such as a cornrootworm would have to develop resistance to both forms of plantprotection, i.e., to the chemical and to the recombinant protein, in thesame generation in order to develop resistance to the combination ofpesticidal agents. Development of resistance under this particularscenario is believed to be highly unlikely, and in fact, virtuallyimpossible. Only root tissue which would grow beyond the zone ofdispersal of the chemical pesticide treatment would be subject to onlyone form of protection. The seed treatment could take the form ofinfusion of chemicals or other compositions into the seed, a coating ofa composition containing various reagents and agents onto the surface ofthe seed, or a capsule that encases the seed and which contains withinit or embedded into the composition of the capsule a compositioncontaining one or more various reagents and agents, chemicals, and thelike which are desired for any number of agriculturally desirablefunction.

In present conventional agricultural practice, in cases where cornfollows corn, it is normal for an insecticide to be applied to protectthe corn root system from severe feeding by rootworm larvae.Conventional practice is to treat for the adult beetles or to treat forthe larvae. Examples of conventional treatment formulations for adultbeetles include the application of carbaryl insecticides (e.g., SEVIN®80S at 1.0-2.0 lbs active/acre); fenvalerate or esfenvalerate (e.g.,PYDRIN® 2.4EC at 0.1 to 0.2 lbs active/acre, or ASANA® 0.66EC at 0.03 to0.05 lbs active/acre); malathion (57% E at 0.9 lbs active/acre);permethrin (e.g., AMBUSH® 2.0EC at 0.1 to 0.2 lbs active/acre, orPOUNCE® 3.2 EC at 0.1 to 0.2 lbs active ingredient/acre); or PENNCAP-M®at 0.25-0.5 lbs active/acre.

To treat for CRW larvae, conventional practice is to apply a soilinsecticide either at or after planting, but preferably as close to egghatching as possible. Conventional treatments include carbofuraninsecticides (e.g., FURADAN® 15G at 8 oz/1000 ft of row); chloropyrifos(e.g., LORSBAN® 15G at 8 oz/1000 ft of row); fonophos (e.g., DYFONATE®20G at 4.5 to 6.0 oz/1000 ft of row); phorate (e.g., THIMET® 20G at 6oz/1000 ft of row); terbufos (e.g., COUNTER® 15G at 8 oz/1000 ft ofrow); or tefluthrin (e.g., FORCE® 3G at 4 to 5 oz/1000 ft of row).

Many of the chemical pesticides listed above are known to be harmful tohumans and to animals in general. The environmental harm that thesepesticides cause is often exacerbated due to the practice of applyingthe pesticides by foliar spraying or direct application to the surfaceof the soil. Wind-drift, leaching, and runoff can cause the migration ofa large fraction of the pesticide out of the desired zone of activityand into surface waters and direct contact with birds, animals andhumans.

Because of concern about the impact of chemical pesticides on publichealth and the health of the environment, significant efforts have beenmade to find ways to reduce the amount of chemical pesticides that areused. Recently, much of this effort has focused on the development oftransgenic crops that are engineered to express insect toxicants derivedfrom microorganisms. For example, U.S. Pat. No. 5,877,012 to Estruch etal. discloses the cloning and expression of proteins from such organismsas Bacillus, Pseudomonas, Clavibacter and Rhizobium into plants toobtain transgenic plants with resistance to such pests as blackcutworms, armyworms, several borers and other insect pests. PublicationWO/EP97/07089 by Privalle et al. teaches the transformation ofmonocotyledons, such as corn, with a recombinant DNA sequence encodingperoxidase for the protection of the plant from feeding by corn borers,earworms and cutworms. Jansens et al., in Crop Sci., 37(5):1616-1624(1997), reported the production of transgenic corn containing a geneencoding a crystalline protein from Bacillus thuringiensis (Bt) thatcontrolled both generations of the European corn borer. A comprehensivereport of field trials of transgenic corn that expresses an insecticidalprotein from B. thuringiensis has been provided by Armstrong et al., inCrop Science, 35(2):550-557 (1995).

It was known that wild-type Bt δ-endotoxins had low activity againstcoleopteran insects, and Kreig et al., in 1983, reported the firstisolation of a coleopteran-toxic B. thuringiensis strain. (See U.S. Pat.No. 4,766,203). U.S. Pat. Nos. 4,797,279 and 4,910,016, also disclosedwild-type and hybrid B. thuringiensis strains that produced proteinshaving some coleopteran activity. More recently, however, amino acidsequence variant forms of Cry3Bb have been developed that havesignificantly higher levels of corn rootworm activity than the activityof the wild type Cry3Bb protein (See, e.g., U.S. Pat. No. 6,023,013,6,060,594, and 6,063,597).

However, it is not known at present whether any transgenic plantexpressing a single insecticide directed to controlling corn rootwormswill be sufficiently effective to protect corn from damage by cornrootworm in heavily infested fields in which crop rotation on an annualbasis is not practiced. In fact, the total control of corn rootwormdamage by any one transgenic event may not be desirable in the longterm, because of the potential for the development of resistant strainsof the target pest.

An alternative to the conventional forms of pesticide or herbicideapplication is the treatment of plant seeds with compositions thatcontain pesticides such as insecticides, nematicides, acaricides,fungicides or with organo-phosphate herbicides, and various forms ofdouble stranded RNA's for use in inhibition of plants pest infestationand the like. The use of fungicides or nematicides to protect seeds, andyoung roots and shoots from attack after planting and sprouting, and theuse of low levels of insecticides for the protection of, for example,corn seed from wireworm, has been used for some time. Seed treatmentwith pesticides has the advantages of providing for the protection ofthe seeds, while minimizing the amount of pesticide required andlimiting the amount of contact with the pesticide and the number ofdifferent field applications necessary to attain control of the pests inthe field. Seed treatment of herbicide resistant naturally occurring ortransgenic varieties with soil stable herbicides that leach into thesoil in effective concentrations for controlling the growth anddevelopment of weeds and parasitic and saprophytic plants within therhizosphere of the crop plant are known in the art.

Other examples of the control of pests by applying insecticides directlyto plant seed are provided in, for example, U.S. Pat. No. 5,696,144,which discloses that the European corn borer caused less feeding damageto corn plants grown from seed treated with a 1-arylpyrazole compound ata rate of 500 g per quintal of seed than control plants grown fromuntreated seed. In addition, U.S. Pat. No. 5,876,739 to Turnblad et al.(and its parent, U.S. Pat. No. 5,849,320) disclose a method forcontrolling soil-borne insects which involves treating seeds with acoating containing one or more polymeric binders and an insecticide.This reference provides a list of insecticides that it identifies ascandidates for use in this coating and also names a number of potentialtarget insects. However, while the U.S. Pat. No. 5,876,739 patent statesthat treating corn seed with a coating containing a particularinsecticide protects corn roots from damage by the corn rootworm, itdoes not indicate or otherwise suggest that such treatment could be usedwith recombinant seed.

The treatment of recombinant seed with nitroimino- ornitroguanidino-compound pesticides has previously been suggested (See,e.g., WO 99/35913), and insecticides such as thiamethoxam, imidacloprid,thiacloprid, and TI-435 (clothianidin) were identified as beingpreferred. In the PCT publication, the use of these insecticides wassuggested for the seeds of a number of different plant species, and forsuch seeds having any one of a long list of potential recombinantinsecticidal traits. However, that reference provided no guidance as tothe details of how such treatments might be effected—such as the amountsof active ingredient that would be necessary per unit amount of seed—andno examples that would give reason to believe that the proposedtreatments would actually provide suitable protection.

Therefore, although recent developments in genetic engineering of plantshave improved the ability to protect plants from pests such as insect,fungal, acaricidal, and nematicidal infestation or from pests such asweeds and parasitic and saprophytic plants, without using chemicalpesticides, and while such techniques as the treatment of seeds withpesticides and herbicides have reduced the harmful effects of pesticidesand herbicides on the environment, numerous problems remain that limitthe successful application of these methods under actual fieldconditions. Accordingly, it would be useful to provide an improvedmethod for the protection of plants, especially corn plants, fromfeeding damage or other detrimental effects caused by pests. It would beparticularly useful if such method would reduce the required applicationrate of conventional chemical pesticides and herbicides, and also if itwould limit the number of separate field operations that were requiredfor crop planting and cultivation.

In addition, it would be useful to have a method of deploying anon-transgenic or transgenic refuge into a field of transgenic cropsinstead of peripheral to a field of transgenic crops according to thepractice presently required by regulatory agencies.

BRIEF SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to a novel methodfor deploying refuge seeds into a field along with seeds from at leastone transgenic crop variety in a mixture by blending together seeds of afirst transgenic crop variety with refuge seeds and ensuring that therefuge and transgenic seeds are blended into a uniform mixture so thatthe blended mixture can be bagged and provided for planting in a field.The transgenic crop seed contains at least one transgene that is notalso present exclusively in any single variety of the refuge seed withwhich the transgenic crop seed is blended, although a part of the refugeseed can contain a transgene that is also present in the transgenic cropseed so long as the percentage of refuge seed not containing thetransgene present in the transgenic crop seed remains at or above alevel that is required by regulatory agencies to establish a refugecomposition. At least from about 100% to about 50% of the blendedmixture consists of the first transgenic crop seed and the balance ofthe mixture is made up of the refuge seeds. The refuge seeds can becomprised of a uniform and single variety of a particular plant species,such as the plant species of which the first transgenic crop consists,or can consist of seeds from a variety of plant species so long as therefuge seeds grow into plants that provide a refuge food source for thepests desired to be controlled by the plants grown from the firsttransgenic plant seeds. The refuge seed can therefore be a crop seedcapable of producing a crop similar to the crop that is capable of beingproduced by the first transgenic crop seeds. The refuge seed can beselected from non-transgenic seed or from a second transgenic seed whichcontains one or more transgenes that are different from those transgenespresent in the first transgenic crop seeds.

The second transgenic seed can be used as all or a part of the refugeseed. The second transgene that is present in the second transgenic seedcan be selected from the group of transgenes consisting of insecticidaltransgenes, fungicidal transgenes, herbicidal transgenes, nematicidaltransgenes, acaricidal transgenes, and bactericidal transgenes. Thefirst transgene can also be selected from this group of transgenes, butshould not be a transgene that is the same as the second transgenepresent in the refuge seeds.

The method provides a means for deploying at least one variety of refugeseed together with at least one variety of transgenic crop seed in afield by blending the seeds together to ensure a uniform mixture andproviding the seeds as a blended mixture or blend for planting in afield. The mixture consists of at least from about 100% to about 50%first transgenic crop seed in comparison to the content percentages ofthe refuge seed. The refuge seed is selected from seed that are eithertransgenic or non-transgenic, but can be a combination of these. In theevent that transgenic refuge seed are selected, the transgenic refugeseed contain a transgene that is different from the transgene present inthe first transgenic crop seed.

A mixture containing from about 100% to about 50% first transgenic cropseed is understood to mean that the composition on either a per weightbasis or a total number of seed basis consists of from about 50% firsttransgenic seed blended together with refuge seed, the composition ofthe refuge seed in the seed blend consisting of from about 50% to about0.01% refuge seed.

The blended seeds mixture can consist of a composition in which thefirst transgenic crop seed has not been coated or treated with a coatingthat contains a seed treatment. Alternatively, the first transgenic cropseed may be coated or treated with a coating that contains a seedtreatment. Similarly, the refuge seeds may be coated or treated with acoating that does not contain a seed treatment or in the alternative maybe coated or treated with a coating that contains a seed treatment. Inany event in which a seed treatment is applied, the seed treatmentshould contain at least one of the agents selected from the groupconsisting of pesticidal agents consisting of but not limited toinsecticides, herbicides, fungicides, nematicides, bactericides, andacaricides.

Insecticides in a seed treatment may consist of any number of agentsincluding but not limited to insecticidal proteins selected from thegroup consisting of recombinant acyl lipid hydrolase protein, a Bacillussphearicus insecticidal protein, Bacillus laterosporous insecticidalprotein, a insecticidal protein derived from a Xenorhabdus bacteriaspecies, a insecticidal protein derived from a Photorhabdus bacteriaspecies, a Bacillus thuringiensis insecticidal δ-endotoxin protein orvegetative insecticidal protein (VIP), and an RNA molecule.

RNA molecules particularly suited for use as insecticidal agents in aseed treatment consist of dsRNAi and sRNAi molecules that act tosuppress gene function in a host or host cell that feeds on the ds orsRNAi molecules present in the seed treatment. The suppression may notresult in death, but may instead result in the stunting, repulsion, orinfertility of the host or host cell that feeds on such molecules.

Insecticides in a seed treatment may also consist of one or morepesticidal agents selected from the group consisting of pyrethrins andsynthetic pyrethroids, oxadizine derivatives, chloronicotinyls,nitroguanidine derivatives, triazoles, organophosphates, pyrrols,pyrazoles, phenyl pyrazoles, diacylhydrazines, biological/fermentationproducts, and carbamates, all as described herein.

The present invention is also directed to a composition comprising oneor more types of refuge plant seeds and at least one type or variety oftransgenic crop seed, blended together into a uniform mixture for use inplanting in a field. The seed blend should be made up of at least onetype of refuge seed, which as described above, can be one or morevarieties or types of refuge seeds, but generally is a uniform varietyor type of refuge seed, and a first transgenic crop seed which containsat least a first transgene that confers some desired benefit upon thecrop (i.e., a benefit such as herbicide tolerance, insect resistance,fungal resistance, nematode resistance, increased yield, decreaseddrought sensitivity, etc.). The first transgenic crop seed should makeup at least from about 100% to about 50% of the blended mixture, withthe balance of the mixture consisting of the refuge seeds. The refugeseeds can be a uniform composition of plant seeds of the same or asimilar variety in relation to the first transgenic crop seeds, and mayalso consist of one or more transgenic varieties of refuge seeds. Therefuge seeds can be selected from non-transgenic varieties of seeds orcan be selected from transgenic varieties, but a transgenic refugevariety should not contain a transgene that is the same as one or moreof the transgenes present in the first transgenic crop seeds.

A seed blend may consist of a composition in which the first transgeniccrop seed has not been coated or treated with a coating that contains aseed treatment. Alternatively, the first transgenic crop seed may becoated or treated with a coating that contains a seed treatment.Similarly, the refuge seeds may be coated or treated with a coating thatdoes not contain a seed treatment or in the alternative may be coated ortreated with a coating that contains a seed treatment. In any event inwhich a seed treatment is applied, the seed treatment should contain atleast one of the agents selected from the group consisting of pesticidalagents consisting of but not limited to insecticides, herbicides,fungicides, nematicides, bactericides, and acaricides.

Insecticides in a seed treatment may consist of any number of agentsincluding but not limited to insecticidal proteins selected from thegroup consisting of recombinant acyl lipid hydrolase protein, a Bacillussphearicus insecticidal protein, Bacillus laterosporous insecticidalprotein, a insecticidal protein derived from a Xenorhabdus bacteriaspecies, a insecticidal protein derived from a Photorhabdus bacteriaspecies, a Bacillus thuringiensis insecticidal δ-endotoxin protein orvegetative insecticidal protein (VIP), and an RNA molecule. RNAmolecules particularly suited for use as insecticidal agents in a seedtreatment consist of dsRNAi and sRNAi molecules that act to suppressgene function in a host or host cell that feeds on the ds or sRNAimolecules present in the seed treatment. The suppression may not resultin death, but may instead result in the stunting, repulsion, orinfertility of the host or host cell that feeds on such molecules.

Insecticides in a seed treatment may also consist of one or morepesticidal agents selected from the group consisting of pyrethrins andsynthetic pyrethroids, oxadizine derivatives, chloronicotinyls,nitroguanidine derivatives, triazoles, organophosphates, pyrrols,pyrazoles, phenyl pyrazoles, diacylhydrazines, biological/fermentationproducts, and carbamates, all as described herein.

The present invention is also directed to a novel seed of a transgeniccorn plant that provides increased resistance to the resulting cornplant against feeding damage by one or more pests, comprising atransgenic event having activity against at least one of the one or morepests, which seed has been treated with an effective amount of apesticide.

The present invention is also directed to a novel transgenic corn seedthat has been treated by the method of the present invention.

The present invention is also directed to a method for deploying anon-transgenic refuge crop into a field of recombinant pest resistantcrops, effectively reducing the labor, costs, and management required todeploy a refuge into a field adjacent to, along side of, or within aclose proximity to a field of recombinant crops. Such a refuge could beeither a mixture of recombinant pest resistant seeds and non-recombinantseeds each treated with a seed coating comprising a chemicalinsecticide, nematicide, herbicide, or fungicide alone or incombination, each present in an amount effective for reducing oreliminating pest infestation within a diffusible zone around the siteinto which the roots of the germinated or sprouted seed wouldintrogress, and through which the root system of the germinated orsprouted seed would grow without inhibition or delay in growth anddevelopment to said root system as a result of the chemical insecticide,nematicide, herbicide, or fungicide composition or coating.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of an improved methodfor the protection of plants, especially corn plants, from damage causedby pests; the provision of such a method that reduces the requiredapplication rate of conventional chemical pesticides includinginsecticides, acaricides, nematicides, herbicides, bactericides andfungicides; also the provision of such a method that limits the numberof separate field operations that are required for crop planting andcultivation; and also the provision of a method for deploying anon-transgenic refuge crop into a field of transgenic crops.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the terms pest, pesticide, and pesticidal are meant tobe interchangeable and inclusive of the following terms: for example,insect, insecticide, and insecticidal when referring to an insect pest;or with the terms, for example, nematode, nematicide, and nematicidalwhen referring to a nematode pest; or with acaric, acaricide, andacaricidal when referring to an acaric pest; or with fungus or fungal,fungicide, and fungicidal or equivalent terms such as mycotic, andmycocidal when referring to fungal or related pests; or with plant orherb, planticide or herbicide, or planticidal or herbicidal whenreferring to a plant or a herb pest or growth and development of weedsand parasitic and saprophytic plants within the rhizosphere of the cropplant.

As used herein, the term “transgenic refuge” refers to the requirementof a resistance management plan for reducing or eliminating thelikelihood of development of resistance to one or more recombinanttraits, such as an insecticide, that is/are either present within arecombinant plant or present adjacent to one or more parts or tissues ofa plant. A refuge may or may not be transgenic, however. Refuge seedsare seeds of plant varieties that grown into plants that act as a refugefor pests that either feed directly on a particular crop species orpests, the presence of which within the local proximity of a particularcrop species, results in the damage, decrease in viability, infertility,or decrease in yield of a crop produced from such crop species.

In accordance with the present invention, it has been discovered thatcorn plants, and by analogy any other plant, can be protected againstfeeding damage by one or more pests by a method that includes providinga transgenic corn seed encoding an insecticidal protein that hasactivity against at least one of the pests and then treating thetransgenic corn seed with an effective amount of a pesticide. Forexample, it has been found that the combination of a transgenic cornseed exhibiting bioactivity against corn rootworm as a result of theproduction of an insecticidal amount of an insecticidal protein withinthe cells of the corn seed and treatment of the seed with certainchemical or protein pesticides provides unexpectedly synergisticadvantages to seeds having such treatment, including unexpectedlysuperior efficacy for protection against damage to the resulting cornplant by corn rootworm. In particular, it is shown herein thattransgenic corn seeds exhibiting bioactivity against corn rootworms as aresult of the production of an amino acid variant of a Cry3Bbδ-endotoxin exhibiting improved insecticidal activity compared to thenative Cry3Bb protein, in combination with the treatment of such seedswith imidacloprid, was unexpectedly superior to either the transgenicevent alone, or to treatment with imidacloprid alone, in protectingresulting corn plants against more severe levels of damage by cornrootworm—levels of damage that are known to reduce corn yield.

Corn plants and seeds that have been engineered to include exogenousgenes derived from Bacillus thuringiensis that encode for the expressionof Cry3 δ-endotoxins having activity against Coleopteran pests areknown, as are methods for the treatment of seeds (even some transgenicseeds) with pesticides. Such useful Cry3 proteins include but are notlimited to Cry3A proteins, Cry3B proteins, and Cry3C proteins. Inaddition, other insecticidal proteins are specifically contemplated tobe effective in the compositions and methods of the present invention.For example, recombinant forms of acyl lipid hydrolases known aspatatins are effective as insecticidal agents (WO 01/49834); and tIC851,CryET70, and Cry22 are effective in controlling corn rootworms (U.S.application Ser. No. 09/853,533 filed May 11, 2001). Also, the binarytoxins CryET33 and CryET34 (WO 98/13498), tIC100 and tIC101 (U.S.Provisional Application Ser. No. 60/232,099 filed Sep. 12, 2000),CryET80 and CryET76 (WO 00/66742), and PS149B1 (Moellenbeck et al.,2001, Nat. Biotechnol. 19:668-672) have all demonstrated corn rootwormcontrolling activity. However, it had not been realized until thepresent invention that certain effective amounts of certain chemical orprotein pesticides could be used to treat recombinant corn seedsexpressing an insecticidal protein, with the result that the combinationwould be unexpectedly superior in increasing the efficacy of both thepesticide and the transgene, and would provide the additional advantagesof increasing the ability to match pesticidal activity against pestpressure, decreasing cost of treatment and/or application, increasingsafety of seed handling, and decreasing environmental impact of eitheror both the event and the pesticide.

In particular, it has been found that the treatment of a transgenic cornseeds that are capable of expressing certain modified Cry3Bb proteinswith from about 100 gm to about 400 gm of certain pesticides per 100 kgof seed provided unexpectedly superior protection against corn rootworm.In addition, it is believed that such combinations are also effective toprotect the emergent corn plants against damage by black cutworm. Theseeds of the present invention are also believed to have the property ofdecreasing the cost of pesticide use, because less of the pesticide canbe used to obtain a required amount of protection than if the innovativemethod is not used. Moreover, because less pesticide is used and becauseit is applied prior to planting and without a separate fieldapplication, it is believed that the subject method is therefore saferto the operator and to the environment, and is potentially lessexpensive than conventional methods.

When it is said that some effects are “synergistic”, it is meant toinclude the synergistic effects of the combination on the pesticidalactivity (or efficacy) of the combination of the transgenic event andthe pesticide. However, it is not intended that such synergistic effectsbe limited to the pesticidal activity, but that they should also includesuch unexpected advantages as increased scope of activity, advantageousactivity profile as related to type and amount of damage reduction,decreased cost of pesticide and application, decreased pesticidedistribution in the environment, decreased pesticide exposure ofpersonnel who produce, handle and plant corn seeds, and other advantagesknown to those skilled in the art.

The present invention also provides an advantage of increasing theability to match pesticidal activity against pest pressure. This refersto the ability to design the combination of the transgenic event and thepesticide treatment so that the seed or the resulting plant is providedwith effective pesticidal activity during the period when feedingpressure from the target pest on the seed or plant reaches its maximum.By way of example, when a pesticide such as imidacloprid or terfluthrinis applied to a corn seed having a corn rootworm transgenic event, thepesticide can be applied in a coating designed to provide controlledrelease of the pesticide. The release rate can be selected so that thepesticide provides protection against such other pests as, for example,black cutworm, at the post emergence stage of corn, while the transgenicevent provides corn rootworm protection at a later stage of plantdevelopment—when such protection is needed.

As used herein, the terms “pesticidal effect” and “pesticidal activity”,or “activity” refer to a toxic effect against a pest. The terms“activity against (one or more) pests”, also have the same meaning. Whenit is said that a seed or plant is “protected against feeding damage byone or more pests”, it is meant that such seed or plant possesses afeature having direct or indirect action on one or more pests thatresults in reduced feeding damage by such pest or pests on the seeds,roots, shoots and foliage of plants having such feature as compared tothe feeding damage caused under the same conditions to plants not havingsuch feature. Such direct or indirect actions include inducing death ofthe pest, repelling the pest from the plant seeds, roots, shoots and/orfoliage, inhibiting feeding of the pest on, or the laying of its eggson, the plant seeds, roots, shoots and/or foliage, and inhibiting orpreventing reproduction of the pest.

The term “insecticidal activity” has the same meaning as pesticidalactivity, except it is limited to those instances where the pest is aninsect. Except where specifically noted, when the term “pesticide” isused herein, that term refers to a chemical pesticide that is suppliedexternally to the seed, and it is not meant to include active agentsthat are produced by the particular seed or the plant that grows fromthe particular seed. However, the terms “pesticidal activity” and“insecticidal activity” can be used with reference to the activity ofeither, or both, an externally supplied pesticide and/or an agent thatis produced by the seed or the plant.

One feature of the present invention is a seed of a transgenic cornplant. As used herein, the terms “transgenic corn plant” mean a cornplant or progeny thereof derived from a transformed corn plant cell orprotoplast, wherein the plant DNA contains an introduced exogenous DNAmolecule not originally present in a native, non-transgenic plant of thesame strain.

It is preferred that the seed contains a gene derived from an exogenoussource, such as a strain of Bacillus thuringiensis, and in particular,it is preferred that the exogenous gene is one that encodes aninsecticidal δ-endotoxin derived from B. thuringiensis. Suchδ-endotoxins are described in WO 99/31248, and include the Cry3 toxins.It is preferred that the δ-endotoxins of the present invention includethe Cry3B proteins, and even more preferred that the δ-endotoxinsinclude the coleopteran-active Cry3Bb proteins. However, as indicatedherein, other insecticidal proteins have been shown to be effective,including but not limited to tIC851, CryET70, Cry22, binary insecticidalproteins CryET33 and CryET34, CryET80 and CryET76, tIC100 and tIC101,and PS 149B1, as well as insecticidal proteins derived from Xenorhabdusand Photorhabdus bacteria species, Bacillus laterosporous species, andBacillus sphearicus species. The nomenclature of the B. thuringiensisinsecticidal crystal proteins was set forth by Höfte and Whitely,Microbiol. Rev.,53:242-255, 1989. This nomenclature has been revised,and the revised nomenclature can be found at Dr. Neil Crickmore'swebsite at the University of Sussex in the United Kingdom atepunix.boils.susx.ac.uk/home/neil-crickmore/bt/index.html. The revisednomenclature will be used herein to describe transgenic event featuresand the δ-endotoxin proteins encoded by the transgenic event. When theterms “transgenic event” are used herein, such terms are meant to referto the genetically engineered DNA that is described above, but also toinclude the protein(s) that are encoded by the modified gene. Atransgenic event in a corn seed, or corn plant, therefore, includes theability to express a protein. When it is said that a “transgenic eventhas activity against a pest”, it is to be understood that it is theprotein that is encoded by the gene that actually has such activity whenthe protein is expressed and brought into contact with the pest.

The term “transgenic event” is also meant herein to include recombinantplants produced by transformation of plant cells with heterologous DNA,i.e., a nucleic acid construct that includes a transgene of interest,regeneration of a population of plants resulting from the insertion ofthe transgene into the genome of the plant, and selection of aparticular plant characterized by insertion into a particular genomelocation. The term “event” refers to the original transformant andprogeny of the transformant that include the heterologous DNA. The term“event” also refers to progeny produced by a sexual outcross between thetransformant and another variety that include the heterologous DNA. Evenafter repeated back-crossing to a recurrent parent, the inserted DNA andflanking DNA from the transformed parent is present in the progeny ofthe cross at the same chromosomal location. The term “event” also refersto DNA from the original transformant comprising the inserted DNA andflanking genomic sequence immediately adjacent to the inserted DNA thatwould be expected to be transferred to a progeny that receives insertedDNA including the transgene of interest as the result of a sexual crossof one parental line that includes the inserted DNA (e.g., the originaltransformant and progeny resulting from selfing) and a parental linethat does not contain the inserted DNA.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison, Wis. (1987).

WO 99/31248 describes methods for genetically engineering B.thuringiensis δ-endotoxin genes so that modified δ-endotoxins can beexpressed. The modified δ-endotoxins differ from the wild-type proteinsby having specific amino acid substitutions, additions or deletions ascompared with the proteins produced by the wild-type organism. Suchmodified δ-endotoxins are identified herein by the use of an asterisk(*), or by reference to a specific protein by its identifying number.Thus, a genetically modified Cry3 δ-endotoxin would be expressed asCry3*, one of which is, for example, Cry3Bb.11231.

Some of the modified δ-endotoxins that are described in WO 99/31248 werefound to have enhanced activity against coleopteran insects, and inparticular against Diabrotica spp., including corn rootworm. As usedherein, the terms “enhanced activity” refer to the increasedinsecticidal activity of a modified toxin as compared with the activityof the same toxin without the amino acid modifications when both aretested under the same conditions. In particular, it was found that Cry3*δ-endotoxins exhibited enhanced activity against corn rootworm, and aretherefore preferred for use in the present invention. More preferred areCry3B* δ-endotoxins, and even more preferred are Cry3Bb* δ-endotoxins.Even more preferred transgenic events are those that comprise theability to express modified δ-endotoxins Cry3Bb.11231 (which wasdeposited on May 27, 1997 as NRRL Accession Number B-21769) andCry3Bb.11098 (which was deposited on Nov. 28, 1997 as NRRL AccessionNumber B-21093). Amino acid sequences for these two preferred proteinsare given in WO 99/31248, as are the nucleotide sequences that encodethem. Transgenic plants known as transgenic events herein derived fromthe insertion of a DNA sequence designed to express the Cry3Bb variantprotein 11231 were designated as transgenic event No. MON853. Transgenicplants known as transgenic events herein derived from the insertion of aDNA sequence designed to express the Cry3Bb variant protein 11098 weredesignated as transgenic event No. MON863.

It has also been found that a preferred use of the present invention isfor reducing pest feeding damage when used in combination with seedshaving transgenic events that have certain levels of effectivenessagainst such pest. To illustrate which levels of effectiveness arepreferred, the following example will use the Iowa Root Rating Method(Hills and Peters, J. Econ. Entomol., 64:764-765, 1971), which measurescorn rootworm feeding damage to corn roots on a 1-6 scale. In therating, 1=no damage or only a few minor feeding scars; 2=feeding scarsevident but no roots eaten off to within 1½ inch of the plant; 3=severalroots eaten off to within 1½ inch of the plant, but never the equivalentof an entire node of roots is destroyed; 4=one root node completelydestroyed; 5=two root nodes completely destroyed; and 6=three or moreroot nodes destroyed. A destroyed root is defined as a root that hasbeen pruned to within 1½ inch of the base. Pruned roots do not have tooriginate from a single node, but all pruned roots must equal theequivalent of a full node to count as a destroyed node.

As used herein, a transgenic event is within the preferred range ofeffectiveness level against a target pest if that event reduces feedingdamage by that pest by a certain amount as compared with the same cropwithout the transgenic event, but does not prevent substantially alldamage by the target pest. For example, if 10% of transgenic cornsuffered corn rootworm damage of 4 or higher on the Iowa 1-6 Scale,while 80% of non-transgenic corn suffered damage of 4 or higher, then itcould be said that the damage to the transgenic corn was(10/80)×100=12.5% of that of the non-transgenic corn. For the purposesof the present invention, it will be understood that a transgenic eventin corn is within the preferred range of effectiveness level if cornhaving such event suffers from about 5% to about 50% of the damagesuffered by non-transgenic corn due to the same pest under the sameconditions. It is more preferred that corn having such transgenic eventsuffers from about 10% to about 40% of the damage suffered bynon-transgenic corn by the same pest under the same conditions, evenmore preferred is damage of from about 15% to about 30%, and yet morepreferred is damage of from about 20% to about 30% of the damagesuffered by non-transgenic corn by the same pest under the sameconditions. As used herein, when the term “about” is used to describethe degree of damage to corn, it is to be understood that the degree ofdamage can be above or below the limits described by as much as 1% or 2%and still be considered to be within the ranges described. By way ofexample, a level of 4.5% damage would be regarded as being “about 5%”.

Without wishing to be bound to this or any other theory, it is believedthat the pesticidal seed treatment can provide significant advantageswhen combined with a transgenic event that provides protection that iswithin the preferred effectiveness range against a target pest. Inaddition, it is believed that there are situations that are well knownto those having skill in the art, where it is advantageous to have suchtransgenic events within the preferred range of effectiveness.

The present invention also includes seeds and plants having more thanone transgenic event. Such combinations are referred to as “stacked”transgenic events. These stacked transgenic events can be events thatare directed at the same target pest, or they can be directed atdifferent target pests. In one preferred method, a seed having theability to express a Cry 3 protein also has the ability to express atleast one other insecticidal protein that is different from a Cry 3protein.

In another preferred method, the seed having the ability to express aCry 3 protein also has a transgenic event that provides herbicidetolerance. It is more preferred that the transgenic event that providesherbicide tolerance is an event that provides resistance to glyphosate,N-(phosphonomethyl)glycine, including the isopropylamine salt form ofsuch herbicide, even more preferred is the transgenic event that iseffective to provide the herbicide resistance of ROUNDUP READY® plantsand seeds available from Monsanto Co., St. Louis, Mo.

In the present method, a corn seed having a transgenic event is treatedwith a pesticide.

Pesticides suitable for use in the invention include pyrethrins andsynthetic pyrethroids; oxadizine derivatives; chloronicotinyls;nitroguanidine derivatives; triazoles; organophosphates; pyrrols;pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentationproducts; and carbamates. Known pesticides within these categories arelisted in The Pesticide Manual, 11th Ed., C. D. S. Tomlin, Ed., BritishCrop Protection Council, Farnham, Surry, UK (1997).

Pyrethroids that are useful in the present composition includepyrethrins and synthetic pyrethroids. The pyrethrins that are preferredfor use in the present method include, without limitation,2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid, and/or(2-methyl-1-propenyl)-2-methoxy-4-oxo-3-(2 propenyl)-2-cyclopenten-1-ylester and mixtures of cis and trans isomers thereof (Chemical AbstractsService Registry Number (“CAS RN”) 8003-34-7).

Synthetic pyrethroids that are preferred for use in the presentinvention include (s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha(1-methylethyl)benzeneacetate (fenvalerate, CAS RN 51630-58-1),(S)-cyano (3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(1-methylethyl)benzeneacetate (esfenvalerate, CAS RN 66230-04-4),(3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylcyclopropane carboxylate (permethrin, CAS RN 52645-53-1), (±)alpha-cyano-(3-phenoxyphenyl)methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate (cypermethrin, CAS RN 52315-07-8), (beta-cypermethrin, CASRN 65731-84-2), (theta cypermethrin, CAS RN 71697-59-1), S-cyano(3-phenoxyphenyl)methyl (±) cis/trans 3-(2,2-dichloroethenyl) 2,2dimethylcyclopropane carboxylate (zeta-cypermethrin, CAS RN 52315-07-8),(s)-alpha-cyano-3-phenoxybenzyl(IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate(deltamethrin, CAS RN 52918-63-5), alpha-cyano-3-phenoxybenzyl2,2,3,3,-tetramethyl cyclopropoanecarboxylate (fenpropathrin, CAS RN64257-84-7),(RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilino]-3-methylbutanoate(tau-fluvalinate, CAS RN 102851-06-9),(2,3,5,6-tetrafluoro-4-methylphenyl)-methyl-(1 alpha, 3alpha)-(Z)-(±)-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate(tefluthrin, CAS RN 79538-32-2), (±)-cyano (3-phenoxyphenyl) methyl(±)-4-(difluoromethoxy)-alpha-(1-methyl ethyl)benzeneacetate(flucythrinate, CAS RN 70124-77-5),cyano(4-fluoro-3-phenoxyphenyl)methyl3-[2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropanecarboxylate(flumethrin, CAS RN 69770-45-2), cyano(4-fluoro-3-phenoxyphenyl)methyl3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate(cyfluthrin, CAS RN 68359-37-5), (beta cyfluthrin, CAS RN 68359-37-5),(transfluthrin, CAS RN 118712-89-3),(S)-alpha-cyano-3-phenoxybenzyl(Z)-(IR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxycarbonyl)vinyl]cyclopropanecarboxylate (acrinathrin, CAS RN 101007-06-1), (IR cis) S and (IS cis) Renantiomer isomer pair ofalpha-cyano-3-phenoxybenzyl-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (alpha-cypermethrin, CAS RN 67375-30-8),[IR,3S)3(1′RS)(1′,2′,2′,2′-tetrabromoethyl)]-2,2-dimethylcyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester(tralomethrin, CAS RN 66841-25-6), cyano-(3-phenoxyphenyl)methyl2,2-dichloro-1-(4-ethoxyphenyl)cyclopropane carboxylate (cycloprothrin,CAS RN 63935-38-6), [1α, 3α(Z)]-(±)-cyano-(3-phenoxyphenyl)methyl3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-cimethylcyclopropanecarboxylate(cyhalothrin, CAS RN 68085-85-8), [1 alpha (s), 3alpha(z)]-cyano(3-phenoxyphenyl)methyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate (lambda cyhalothrin, CAS RN 91465-08-6), (2-methyl[1,1′-biphenyl]-3-yl)methyl3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cyclopropanecarboxylate(bifenthrin, CAS RN 82657-04-3),5-1-benzyl-3-furylmethyl-d-cis(1R,3S,E)2,2-dimethyl-3-(2-oxo,-2,2,4,5tetrahydrothiophenylidenemethyl)cyclopropane carboxylate (kadethrin,RU15525, CAS RN 58769-20-3), [5-(phenyl methyl)-3-furanyl]-3-furanyl2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylate(resmethrin, CAS RN 10453-86-8),(1R-trans)-[5-(phenylmethyl)-3-furanyl]methyl2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropanecarboxylate(bioresmethrin, CAS RN 28434-01-7), 3,4,5,6-tetrahydro-phthalimidomethyl-(IRS)-cis-trans-chrysanthemate (tetramethrin,CAS RN 7696-12-0), 3-phenoxybenzyl-d,l-cis,trans2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate (phenothrin,CAS RN 26002-80-2); (empenthrin, CAS RN 54406-48-3); (cyphenothrin; CASRN 39515-40-7), (prallethrin, CAS RN 23031-36-9), (imiprothrin, CAS RN72963-72-5), (RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(1A,3R;1R,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropane carboxylate(allethrin, CAS RN 584-79-2), (bioallethrin, CAS RN 584-79-2), and(ZXI8901, CAS RN 160791-64-0). It is believed that mixtures of one ormore of the aforementioned synthetic pyrethroids can also be used in thepresent invention. Particularly preferred synthetic pyrethroids aretefluthrin, lambda cyhalothrin, bifenthrin, permethrin and cyfluthrin.Even more preferred synthetic pyrethroids are tefluthrin and lambdacyhalothrin, and yet more preferred is tefluthrin.

Insecticides that are oxadiazine derivatives are useful in the subjectmethod. The oxadizine derivatives that are preferred for use in thepresent invention are those that are identified in U.S. Pat. No.5,852,012. More preferred oxadiazine derivatives are5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine,5-(2-chlorothiazol-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine,3-methyl-4-nitroimino-5-(1-oxido-3-pyridinomethyl)perhydro-1,3,5-oxadiazine,5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxidiazine;and3-methyl-5-(2-methylpyrid-5-ylmethyl)-4-nitroiminoperhydro-1,3,5-oxadiazine.Even more preferred is thiamethoxam (CAS RN 153719-23-4).

Chloronicotinyl insecticides are also useful in the subject method.Chloronicotinyls that are preferred for use in the subject compositionare described in U.S. Pat. No. 5,952,358, and include acetamiprid((E)-N-[(δ-chloro-3-pyridinyl)methyl]-N′-cyano-N-methyleneimidamide, CASRN 135410-20-7),imidacloprid(1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2-imidazolidinimime,CAS RN 138261-41-3), andnitenpyram(N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N′-methyl-2-nitro-1,1-ethenediamine,CAS RN 120738-89-8).

Nitroguanidine insecticides are useful in the present method. Suchnitroguanidines can include those described in U.S. Pat. Nos. 5,633,375,5,034,404 and 5,245,040.

Pyrrols, pyrazoles and phenyl pyrazoles that are useful in the presentmethod include those that are described in U.S. Pat. No. 5,952,358.Preferred pyrazoles includechlorfenapyr(4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethylpyrrole-3-carbonitrile,CAS RN 122453-73-0), fenpyroximate((E)-1,1-dimethylethyl-4[[[[(1,3-dimethyl-5-phenoxy-1H-pyrazole-4-yl)methylene]amino]oxy]methyl]benzoate,CAS RN 111812-58-9), and tebufenpyrad(4-chloro-N[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide,CAS RN 119168-77-3). A preferred phenyl pyrazole is fipronil(5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1R,S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile,CAS RN 120068-37-3).

Diacylhydrazines that are useful in the present invention includehalofenozide(4-chlorobenzoate-2-benzoyl-2-(1,1-dimethylethyl)-hydrazide, CAS RN112226-61-6), methoxyfenozide (RH-2485;N-tert-butyl-N′-(3-methoxy-o-toluoyl)-3,5-xylohydrazide, CAS RN161050-58-4), and tebufenozide (3,5-dimethylbenzoic acid1-(1,1-dimethylethyl)-2,(4-ethylbenzoyl)hydrazide, CAS RN 112410-23-8).

Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate are usefulin the nethod of the present invention. A preferred triazole istriazamate (ethyl[[1-[(dimethylamino)carbonyl]-3-(1,1-dimethylethyl)-1H-1,2,4-triazol-5-yl]thio]acetate,CAS RN 112143-82-5).

Biological/fermentation products, such as avermectin (abamectin, CAS RN71751-41-2) and spinosad (XDE-105, CAS RN 131929-60-7) are useful in thepresent method.

Organophosphate insecticides are also useful as one of the components ofthe present method. Preferred organophophate insecticides includeacephate (CAS RN 30560-19-1), chlorpyrifos (CAS RN 2921-88-2),chlorpyrifos-methyl (CAS RN 5598-13-0), DIAZINON(diethoxy-(δ-methyl-2-propan-2-ylpyrimidin-4-yl)oxy-sulfanylidenephosphorane)(CAS RN 333-41-5), fenamiphos (CAS RN 22224-92-6), and MALATHION(diethyl 2-dimethoxyphosphinothioylsulfanylbutanedioate) (CAS RN121-75-5).

In addition, carbamate insecticides are useful in the subject method.Preferred carbamate insecticides are aldicarb (CAS RN 116-06-3),carbaryl (CAS RN 63-25-2), carbofuran (CAS RN 1563-66-2), oxamyl (CAS RN23135-22-0) and thiodicarb (CAS RN 59669-26-0).

When an insecticide is described herein, it is to be understood that thedescription is intended to include salt forms of the insecticide as wellas any isomeric and/or tautomeric form of the insecticide that exhibitsthe same insecticidal activity as the form of the insecticide that isdescribed.

The insecticides that are useful in the present method can be of anygrade or purity that pass in the trade as such insecticide. Othermaterials that accompany the insecticides in commercial preparations asimpurities can be tolerated in the subject methods and compositions, aslong as such other materials do not destabilize the composition orsignificantly reduce or destroy the activity of any of the insecticidecomponents or the transgenic event against the target pest(s). One ofordinary skill in the art of the production of insecticides can readilyidentify those impurities that can be tolerated and those that cannot.

It has been found that the present method is useful to protect seeds andplants against a wide array of agricultural pests, including insects,mites, fungi, yeasts, molds, bacteria, nematodes, weeds, and parasiticand saprophytic plants.

When an insect is the target pest for the present invention, such pestsinclude but are not limited to:

from the order Lepidoptera, for example,

Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabamaargillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp,Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella,Carposina nipponensis, Chilo spp., Choristoneura spp., Clysiaambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp.,Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydiaspp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp.,Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp.,Ostrinia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea,Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp.,Tortrix spp., Trichoplusia ni and Yponomeuta spp.;

from the order Coleoptera, for example,

Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp.;

from the order Orthoptera, for example,

Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae,Locusta spp., Periplaneta ssp., and Schistocerca spp.;

from the order Isoptera, for example,

Reticulitemes ssp;

from the order Psocoptera, for example,

Liposcelis spp.;

from the order Anoplura, for example,

Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. andPhylloxera spp.;

from the order Mallophaga, for example,

Damalinea spp. and Trichodectes spp.;

from the order Thysanoptera, for example,

Franklinella spp., Hercinothrips spp., Taeniothrips spp., Thrips palmi,Thrips tabaci and Scirtothrips aurantii;

from the order Heteroptera, for example,

Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp.,Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodniusspp., Sahlbergella singularis, Scotinophara spp. and Triatoma spp.;

from the order Homoptera, for example,

Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri;

from the order Hymenoptera, for example,

Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpiniapolytoma, Hoplocampa spp., Lasius sppp., Monomoriurn pharaonis,Neodiprion spp, Solenopsis spp. and Vespa ssp.;

from the order Diptera, for example,

Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphoraerythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebraspp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilusspp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp.,Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseoliaspp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletispomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. andTipula spp.,

from the order Siphonaptera, for example,

Ceratophyllus spp. and Xenopsylla cheopis and

from the order Thysanura, for example,

Lepisma saccharina.

It has been found that the present invention is particularly effectivewhen the insect pest is a Diabrotica spp., and especially when the pestis Diabrotica virgifera, Diabrotica barberi, or Diabroticaundecimpunctata.

Another application wherein the present invention is believed to beparticularly effective is when the pesticide has activity against a weedor a parasitic or saprophytic plant and the transgenic event hasactivity against a member selected from the group consisting ofDiabrotica virgifera, Diabrotica barberi and Diabrotica undecimpunctata.This is believed to be more preferred useful when the weed or aparasitic or saprophytic plant is the African plant known as “Striga”,and even more preferred when the pesticide is ROUNDUP® (available fromMonsanto Company).

In the method of the present invention, the pesticide is applied to atransgenic corn seed. Although it is believed that the present methodcan be applied to a transgenic corn seed in any physiological state, itis preferred that the seed be in a sufficiently durable state that itincurs no damage during the treatment process. Typically, the seed wouldbe a seed that had been harvested from the field; removed from theplant; and separated from any cob, stalk, outer husk, and surroundingpulp or other non-seed plant material. The seed would preferably also bebiologically stable to the extent that the treatment would cause nobiological damage to the seed. In one embodiment, for example, thetreatment can be applied to seed corn that has been harvested, cleanedand dried to a moisture content below about 15% by weight. In analternative embodiment, the seed can be one that has been dried and thenprimed with water and/or another material and then re-dried before orduring the treatment with the pesticide. Within the limitations justdescribed, it is believed that the treatment can be applied to the seedat any time between harvest of the seed and sowing of the seed. As usedherein, the term “unsown seed” is meant to include seed at any periodbetween the harvest of the seed and the sowing of the seed in the groundfor the purpose of germination and growth of the plant.

When it is said that unsown seed is “treated” with the pesticide, suchtreatment is not meant to include those practices in which the pesticideis applied to the soil, rather than to the seed. For example, suchtreatments as the application of the pesticide in bands, “T”-bands, orin-furrow, at the same time as the seed is sowed are not considered tobe included in the present invention.

The pesticide, or combination of pesticides, can be applied “neat”, thatis, without any diluting or additional components present. However, thepesticide is typically applied to the seeds in the form of a pesticideformulation. This formulation may contain one or more other desirablecomponents including but not limited to liquid diluents, binders toserve as a matrix for the pesticide, fillers for protecting the seedsduring stress conditions, and plasticizers to improve flexibility,adhesion and/or spreadability of the coating. In addition, for oilypesticide formulations containing little or no filler, it may bedesirable to add to the formulation drying agents such as calciumcarbonate, kaolin or bentonite clay, perlite, diatomaceous earth or anyother adsorbent material. Use of such components in seed treatments isknown in the art. See, e.g., U.S. Pat. No. 5,876,739. The skilledartisan can readily select desirable components to use in the pesticideformulation depending on the seed type to be treated and the particularpesticide that is selected. In addition, readily available commercialformulations of known pesticides may be used, as demonstrated in theexamples below.

The seeds may also be treated with one or more of the followingingredients: other pesticides, including compounds which act only belowthe ground; fungicides, such as captan, thiram, metalaxyl,(methoxam=resolved isomer of metalaxyl), fludioxonil, oxadixyl, andisomers of each of those materials, and the like; herbicides, includingcompounds selected from carbamates, thiocarbamates, acetamides,triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils,phenoxys, ureas, and benzoic acids; herbicidal safeners such asbenzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide,various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone,naphthalic anhydride compounds, and oxime derivatives; fertilizers;biocontrol agents such as naturally-occurring or recombinant bacteriaand fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia,Trichoderma, Glomus, Gliocladium and mycorrhizal fungi; and doublestranded or stabilized RNA molecules that, when consumed by a pest orpest cell, act to reduce or eliminate some essential function within thepest or pest cell which further results in one or more of the death,infertility, feeding suppression, stunting, repulsion, or lack offurther development and differentiation of the pest or pest cell. Theseingredients may be added as a separate layer on the seed oralternatively may be added as part of the pesticide composition.

Preferably, the amount of the novel composition or other ingredientsused in the seed treatment should not inhibit generation of the seed, orcause phytotoxic damage to the seed.

The pesticide formulation that is used to treat the transgenic corn seedin the present invention can be in the form of a suspension; emulsion;slurry of particles in an aqueous medium (e.g., water); wettable powder;wettable granules (dry flowable); and dry granules. If formulated as asuspension or slurry, the concentration of the active ingredient in theformulation is preferably about 0.5% to about 99% by weight (w/w),preferably 5-40%.

As mentioned above, other conventional inactive or inert ingredients canbe incorporated into the formulation. Such inert ingredients include butare not limited to: conventional sticking agents, dispersing agents suchas methylcellulose (METHOCEL A15LV or METHOCEL A15C, for example, serveas combined dispersant/sticking agents for use in seed treatments),polyvinyl alcohol (e.g., ELVANOL 51-05), lecithin (e.g., YELKINOL P),polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVP/VAS-630), thickeners (e.g., clay thickeners such as VAN GEL B to improveviscosity and reduce settling of particle suspensions), emulsionstabilizers, surfactants, antifreeze compounds (e.g., urea), dyes,colorants, and the like. Further inert ingredients useful in the presentinvention can be found in McCutcheon's, vol. 1, “Emulsifiers andDetergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.Additional inert ingredients useful in the present invention can befound in McCutcheon's, vol. 2, “Functional Materials,” MC PublishingCompany, Glen Rock, N.J., U.S.A., 1996.

The pesticides and pesticide formulations of the present invention canbe applied to seeds by any standard seed treatment methodology,including but not limited to mixing in a container (e.g., a bottle orbag), mechanical application, tumbling, spraying, and immersion. Anyconventional active or inert material can be used for contacting seedswith pesticides according to the present invention, such as conventionalfilm-coating materials including but not limited to water-based filmcoating materials such as Sepiret (Seppic, Inc., Fairfield, N.J.) andOpacoat (Berwind Pharm. Services, Westpoint, Pa.).

The subject pesticides can be applied to a seed as a component of a seedcoating.

Seed coating methods and compositions that are known in the art areuseful when they are modified by the addition of one of the embodimentsof the combination of pesticides of the present invention. Such coatingmethods and apparatus for their application are disclosed in, forexample, U.S. Pat. Nos. 5,918,413, 5,891,246, 5,554,445, 5,389,399,5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coating compositionsare disclosed, for example, in U.S. Pat. Nos. 5,939,356, 5,882,713,5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103, 5,622,003,5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587, 4,383,391,4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.

Useful seed coatings contain one or more binders and at least one of thesubject combinations of pesticides.

Binders that are useful in the present invention preferably comprise anadhesive polymer that may be natural or synthetic and is withoutphytotoxic effect on the seed to be coated. The binder may be selectedfrom polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinylacetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; fats; oils; proteins, including gelatin and zeins; gumarabics; shellacs; vinylidene chloride and vinylidene chloridecopolymers; calcium lignosulfonates; acrylic copolymers;polyvinylacrylates; polyethylene oxide; acrylamide polymers andcopolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; andpolychloroprene.

It is preferred that the binder be selected so that it can serve as amatrix for the subject pesticides. While the binders disclosed above mayall be useful as a matrix, the specific binder will depend upon theproperties of the combination of pesticides. The term “matrix”, as usedherein, means a continuous solid phase of one or more binder compoundsthroughout which is distributed as a discontinuous phase one or more ofthe subject pesticides. Optionally, a filler and/or other components canalso be present in the matrix. The term matrix is to be understood toinclude what may be viewed as a matrix system, a reservoir system or amicroencapsulated system. In general, a matrix system consists ofpesticides of the present invention and filler uniformly dispersedwithin a polymer, while a reservoir system consists of a separate phasecomprising the subject pesticides, that is physically dispersed within asurrounding, rate-limiting, polymeric phase. Microencapsulation includesthe coating of small particles or droplets of liquid, but also todispersions in a solid matrix.

The amount of binder in the coating can vary, but will be in the rangeof about 0.01 to about 25% of the weight of the seed, more preferablyfrom about 0.05 to about 15%, and even more preferably from about 0.1%to about 10%.

As mentioned above, the matrix can optionally include a filler. Thefiller can be an absorbent or an inert filler, such as are known in theart, and may include woodflours, clays, activated carbon, sugars,diatomaceous earth, cereal flours, fine-grain inorganic solids, calciumcarbonate, and the like. Clays and inorganic solids, which may be used,include calcium bentonite, kaolin, china clay, talc, perlite, mica,vermiculite, silicas, quartz powder, montmorillonite and mixturesthereof. Sugars, which may be useful, include dextrin and maltodextrin.Cereal flours include wheat flour, oat flour and barley flour.

The filler is selected so that it will provide a proper microclimate forthe seed, for example the filler is used to increase the loading rate ofthe active ingredients and to adjust the control-release of the activeingredients. The filler can aid in the production or process of coatingthe seed. The amount of filler can vary, but generally the weight of thefiller components will be in the range of about 0.05 to about 75% of theseed weight, more preferably about 0.1 to about 50%, and even morepreferably about 0.5% to 15%.

The pesticides that are useful in the coating are those pesticides thatare described herein. The amount of pesticide that is used for thetreatment of the seed will vary depending upon the type of seed and thetype of active ingredients, but the treatment will comprise contactingthe seeds with an amount of the combination of pesticides that ispesticidally effective. When insects are the target pest, that amountwill be an amount of the insecticide that is insecticidally effective.As used herein, an insecticidally effective amount means that amount ofinsecticide that will kill insect pests in the larvae or pupal state ofgrowth, or will consistently reduce or retard the amount of damageproduced by insect pests.

In general, the amount of pesticide that is applied to the seed in thetreatment will range from about 10 gm to about 2000 gm of the activeingredient of the pesticide per 100 kg of the weight of the seed.Preferably, the amount of pesticide will be within the range of about 50gm to about 1000 gm active per 100 kg of seed, more preferably withinthe range of about 100 gm to about 600 gm active per 100 kg of seed, andeven more preferably within the range of about 200 gm to about 500 gm ofactive per 100 kg of seed weight. Alternatively, it has been found to bepreferred that the amount of the pesticide be over about 60 gm of theactive ingredient of the pesticide per 100 kg of the seed, and morepreferably over about 80 gm per 100 kg of seed.

In preferred embodiments of the present invention the transgenic eventcomprises the ability to express a Cry3Bb.11231 protein or aCry3Bb.11098 protein, and the pesticide is selected from eitherimidacloprid or tefluthrin.

The pesticides that are used in the treatment must not inhibitgermination of the seed and should be efficacious in protecting the seedand/or the plant during that time in the target insect's life cycle inwhich it causes injury to the seed or plant. In general, the coatingwill be efficacious for approximately 0 to 120 days after sowing.

The pesticides of the subject invention can be applied to the seed inthe form of a coating. The use of a coating is particularly effective inaccommodating high pesticidal loads, as can be required to treattypically refractory pests, such as corn rootworm, while at the sametime preventing unacceptable phytotoxicity due to the increasedpesticidal load.

Optionally, a plasticizer can be used in the coating formulation.Plasticizers are typically used to make the film that is formed by thecoating layer more flexible, to improve adhesion and spreadability, andto improve the speed of processing. Improved film flexibility isimportant to minimize chipping, breakage or flaking during storage,handling or sowing processes. Many plasticizers may be used, however,useful plasticizers include polyethylene glycol, glycerol,butylbenzylphthalate, glycol benzoates and related compounds. The rangeof plasticizer in the coating layer will be in the range of from bout0.1 to about 20% by weight.

When the pesticide used in the coating is an oily type formulation andlittle or no filler is present, it may be useful to hasten the dryingprocess by drying the formulation. This optional step may beaccomplished by means will known in the art and can include the additionof calcium carbonate, kaolin or bentonite clay, perlite, diatomaceousearth, or any absorbent material that is added preferably concurrentlywith the pesticidal coating layer to absorb the oil or excess moisture.The amount of calcium carbonate or related compounds necessary toeffectively provide a dry coating will be in the range of about 0.5 toabout 10% of the weight of the seed.

The coatings formed with the pesticide are preferably of the type thatare capable of effecting a slow rate of release of the pesticide bydiffusion or movement through the matrix to the surrounding medium.

In addition to the coating layer, the seed may be treated with one ormore of the following ingredients: other pesticides including fungicidesand herbicides; herbicidal safeners; fertilizers and/or biocontrolagents. These ingredients may be added as a separate layer oralternatively may be added in the pesticidal coating layer.

The pesticide formulation may be applied to the seeds using conventionalcoating techniques and machines, such as fluidized bed techniques, theroller mill method, rotostatic seed treaters, and drum coaters. Othermethods, such as spouted beds may also be useful. The seeds may bepresized before coating. After coating, the seeds are typically driedand then transferred to a sizing machine for sizing. Such procedures areknown in the art.

The pesticide-treated seeds may also be enveloped with a filmovercoating to protect the pesticide coating. Such overcoatings areknown in the art and may be applied using conventional fluidized bed anddrum film coating techniques.

In another embodiment of the present invention, a pesticide can beintroduced onto or into a seed by use of solid matrix priming. Forexample, a quantity of the pesticide can be mixed with a solid matrixmaterial and then the seed can be placed into contact with the solidmatrix material for a period to allow the pesticide to be introduced tothe seed. The seed can then optionally be separated from the solidmatrix material and stored or used, or the mixture of solid matrixmaterial plus seed can be stored or planted directly. Solid matrixmaterials which are useful in the present invention includepolyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea,polyacrylate, or any other material capable of absorbing or adsorbingthe pesticide for a time and releasing that pesticide into or onto theseed. It is useful to make sure that the pesticide and the solid matrixmaterial are compatible with each other. For example, the solid matrixmaterial should be chosen so that it can release the pesticide at areasonable rate, for example over a period of minutes, hours, or days.

The present invention further embodies imbibition as another method oftreating seed with the pesticide. For example, plant seed can becombined for a period of time with a solution comprising from about 1%by weight to about 75% by weight of the pesticide in a solvent such aswater. Preferably the concentration of the solution is from about 5% byweight to about 50% by weight, more preferably from about 10% by weightto about 25% by weight. During the period that the seed is combined withthe solution, the seed takes up (imbibes) a portion of the pesticide.Optionally, the mixture of plant seed and solution can be agitated, forexample by shaking, rolling, tumbling, or other means. After imbibition,the seed can be separated from the solution and optionally dried, forexample by patting or air drying.

In yet another embodiment, a powdered pesticide can be mixed directlywith seed. Optionally, a sticking agent can be used to adhere the powderto the seed surface. For example, a quantity of seed can be mixed with asticking agent and optionally agitated to encourage uniform coating ofthe seed with the sticking agent. The seed coated with the stickingagent can then be mixed with the powdered pesticide. The mixture can beagitated, for example by tumbling, to encourage contact of the stickingagent with the powdered pesticide, thereby causing the powderedpesticide to stick to the seed.

The present invention also provides a transgenic corn seed that has beentreated with a pesticide by the method described above.

The treated seeds of the present invention can be used for thepropagation of corn plants in the same manner as conventional treatedcorn seed. The treated seeds can be stored, handled, sowed and tilled inthe same manner as any other pesticide treated seed. Appropriate safetymeasures should be taken to limit contact of the treated seed withhumans, food or feed materials, water and birds and wild or domesticanimals.

In a preferred embodiment, the invention is an insect transgenic seedmix refuge strategy, i.e., 10% non-transgenic seed, combined with aninsecticidal seed treatment. The combination of seed mix refuge strategyin combination with a seed treatment allows for protection of thenon-transgenic plants in the mixture and provides a second mode ofaction for the transgenic seeds. The combination refuge strategy andsecond mode of action are optimal in delaying the onset of resistancedevelopment. This assumes that larvae would survive to adults on thenon-transgenic plants and at the same time that these plants aresufficiently protected by the seed treatment. The seed treatment may beon all seed or only on the non-transgenic seed within the mix.

The words “seed mix refuge strategy” is intended to refer to a means fordeploying into a field of crops some percentage of the seeds whichsprout and develop into mature refuge plants but do not contain atransgene that is present in the crop seeds, thus allowing susceptibleadults to survive. Although this strategy may be acceptable on low tomoderate levels of insect pressure, under very high levels of insectpressure the non-protected plants, i.e. refuge plants, may be damagedsuch that this insect resistance management strategy is not commerciallyviable. By combining the mix seed refuge strategy with a seed treatment,the refuge plants are sufficiently protected but still allow for larvalsurvivorship to adults, and the seed mix refuge strategy becomescommercially viable under all levels of insect pressure. At the sametime, two modes of action are achieved, assuring the longest possibleterm for commercial viability and utility of the transgenic crop seedswith a minimal risk to the development of resistance races of insects.

One alternative to the approaches outlined above for achieving aseed-mix or seed-blend refuge composition would be to select a firsttransgenic crop seed to be mixed with a refuge seed to form a seedblend. The seed blend consists at least of a first transgenic crop seed,which contains at least a first transgene, and at least one type ofrefuge plant seed. The refuge plant seed can be uniform in nature, inthat it is composed of a single type of seed from a single variety ofplant, or can be non-uniform in nature and consist of two or morevarieties of plant. Preferably the refuge seed is similar in variety tothe first transgenic crop seed. The refuge seed can be non-transgenic orcan be transgenic. A refuge seed that is a transgenic seed can containany transgene so long as it is not a transgene that is present in thefirst transgenic crop seed. It is preferable that a transgene in atransgenic refuge seed be a transgene selected from an insecticidalgene, a herbicide tolerance gene, a fungicide tolerance gene, and thelike.

In the regulatory environment that currently exists today, obtaining theapproval of an appropriate regulatory agency for commercialization of arecombinant plant generally requires that a percentage of all of thecrop that is planted by a particular farmer intending to plant a cropcontaining a recombinant trait which effects the survival of particularinsect pests be planted as a refuge of non-recombinant or non-transgeniccrops, or crops which do not contain the ability to inhibit thedevelopment and growth of the particular insect pest by the same mode ofaction. In fact, it is preferred by the regulatory agencies that therefuge crop be planted with a non-transgenic crop, and it is furtherrequired that the refuge be planted as a block separate and apart fromthe recombinant crops. In addition, the percentage of the total cropplanted is required to be at least 1% refuge, more preferably betweenfrom about 2 to about 5% refuge, even more preferably between from about5% to about 10% refuge, and more preferably still between from about 10%to about 20% refuge or more depending on the amount of insect pressureexpected for a particular geographic location and depending also on thetype of crop plant subject to regulatory requirements. Such practicescause added expense for farmers in terms of their input into labor andfinancial expenses, and are difficult to police. Even though farmers arerequired to purchase enough non-recombinant seed to plant the requiredrefuge along with any recombinant seed purchase, the added labor forplanting and segregating the refuge and the likely lower yields withinthe refuge as a result of greater insect infestation is a disincentivefor the farmer to comply with the regulatory requirements. Thus, a seedmix containing the requisite refuge amount of non-transgenic seed, andwhich is treated with an insecticide to protect the refuge plants frominfestation, would be a commercially acceptable means for ensuringcompliance with regulatory agency refuge strategies.

Advantages of a seed mix deployable refuge strategy over a block refugestrategy includes elimination of the issues around enforcement andcompliance, simplicity, and complementarity with block refuge strategiesrequired for other insect resistance traits. Furthermore by adding aseed treatment to the seed mix deployable refuge strategy, no plants areleft unprotected in the field and a second mode of action is uniformlyintroduced to function along with the transgenic insect control means.

The seed mix deployable transgenic refuge strategy is particularlysignificant for corn rootworm resistant transgenic corn, for which aseed mix refuge strategy may be the only feasible means of deploying arefuge for the production of susceptible corn rootworms that will matewith any resistant individuals which may survive upon feeding on a cornrootworm resistant transgenic plant. By combining a seed treatment withthe corn rootworm transgenic and non-transgenic seed in a mix, the seedmix refuge strategy would then be commercially viable, because thenon-transgenic seed would be sufficiently protected by the seedtreatment and still allow for sufficient numbers of larvae to survive toadults while continuing to provide for susceptible adult insectsemerging from the field of crops.

This invention eliminates the necessity for grower application ofchemical or other insecticides to the refuge to protect the plants aswould be the case in a block refuge strategy. In the absence of seedtreatment on the transgenic seeds in such a mix, the transgenic seedssprout and send their roots outward and downward. Target insects whichfeed on these roots necessarily succumb to the levels of theinsecticidal protein preferentially produced in the root tissue of theplant. In this scenario, the seeds comprising the non-transgenic refugemixed uniformly into the seed mix deployable refuge mixture can eitherbe treated with a chemical insecticide or left untreated. Of course theuntreated refuge seed in the mixture would be entirely susceptible toinsect infestation, generally resulting in a yield loss with respect tothe percentage of refuge seed contained within the mixture. Ideally,however, the refuge seed would be treated with a composition whichcontains at least one and perhaps two or more insecticidal agentsselected from the group consisting of chemical insecticide andbiologically derived insecticidal agents such as Bacillus thuringiensisinsecticidal δ-endotoxin protein or vegetative insecticidal protein(VIP), Bacillus sphearicus insecticidal protein, Bacillus laterosporousinsecticidal protein, insecticidal proteins derived from Xenorhabdus andPhotorhabdus bacteria species, and insecticidal proteins which have beenspecifically demonstrated to be effective, including but not limited totIC851, CryET70, Cry22, and binary insecticidal proteins CryET33 andCryET34, CryET80 and CryET76, tIC100 and tIC 101, and PS149B1. Treatedrefuge seeds within the mix would sprout when planted and the rootswould grow outward and downward away from the soil surface. Commensuratewith planting and exposure to the moisture in the soil, the treatmentcomposition on the seed would disperse into the microenvironment of theseed in the soil, providing a decreasing concentration of insecticidalagent as a second mode of action through which the young root tissuewould have to extend in order to be susceptible to insect feeding.Ordinarily, the microenvironment into which the insecticidal agentswould disperse would be from about 1 to about 5 centimeters from thepoint of dispersion, or from about 1 to about 10 centimeters from thepoint of dispersion, said point of dispersion being defined as thecenterpoint of the seed mass within the soil at the time of germination.Ordinarily, an insecticidally effective dose of the chemical or proteinagent contained within the seed treatment would be required to extendoutward for some distance from the centerpoint of seed mass within thesoil at the time of germination. That effective dose would be requiredto be within the dispersal zone around the seed mass, generally beingfrom about 1 to about 5 centimeters from the point of dispersion, andmore preferably from about 1 to about 10 centimeters from the point ofdispersion, and even more preferably from about 2 to about 10centimeters from the point of dispersion.

The more preferable means of deploying a transgenic refuge into a fieldof recombinant crops would comprise a seed mixture comprising from about1% to about 10% refuge seed or more preferably from about 1 to about 20%refuge seed. This embodiment encompasses the treatment of all seedscontained within the mixture, such that the afore mentioned dispersalzone around the center of mass of any of the seeds planted into the soilwould suffice. It is also envisioned that regulatory requirements wouldmandate a refuge requirement greater than the aforementioned 20%, and itis intended that those greater requirements for refuge be includedwithin the scope of this invention.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

The following examples describe preferred embodiments of the invention.Other embodiments within the scope of the claims herein will be apparentto one skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by the claimswhich follow the examples. In the examples all percentages are given ona weight basis unless otherwise indicated.

EXAMPLE 1

Production of transgenic corn seed effective against corn rootworm andtreatment of such seed with imidacloprid (Gaucho®) and tefluthrin(Raze®).

Corn seeds were prepared to express amino acid sequence variant proteinsof a Coleopteran inhibitory Bacillus thuringiensis Cry3Bb δ-endotoxin(Cry3Bb.11231 (MON853) or Cry3Bb.11098 (MON863)) by the methodsdescribed for these respective events in WO 99/31248. Such variantproteins have been shown to exhibit improved levels of bioactivity incontrolling pests such as Diabrotica species. (U.S. Pat. No. 6,063,597).

Corn transformation event MON853 contains a nucleotide sequence whichhas not been optimized for plant expression. The insecticidal Cry3Bbprotein variant produced by the MON853 event has been shown to exhibitimproved insecticidal activity, in particular directed against cornrootworms. While it is not preferred that a nucleotide sequence encodingan insecticidal protein from Bacillus thuringiensis be introduced into aplant without first being modified to remove sequences which cause theresulting protein to be produced inefficiently, it is believed that thecoding sequence within event MON853 functions to produce effectiveinsecticidal activity in part because the length of the amino acidsequence which comprises a Cry3Bb variant protein is about half of whata lepidopteran effective insecticidal Bacillus thuringiensis Cryprotein, and because the MON853 variant protein, Cry3Bb.11231 has fromabout 3 to about 10 fold greater bioactivity against corn rootworms thanthe native Cry3Bb protein derived from Bacillus thuringiensis. NativeBacillus thuringiensis nucleotide sequences encoding truncated Cryproteins exhibiting lepidopteran inhibitory bioactivity are about thesame size as the sequence encoding Cry3Bb variants exemplified in theseexamples herein, and have been shown to be expressed at very low butineffective levels in some plants.

Corn transformation event MON863 contains a modified nucleotide sequencewhich has been optimized for plant expression. The insecticidal Cry3Bbprotein variant produced by the MON863 event, designated Cry3Bb.11098,has been shown to exhibit improved insecticidal activity, in particulardirected against corn rootworms. MON863 exhibits better corn rootwormcontrol than MON853 with or without seed treatment, more likely than notbecause the MON863 event contains a modified sequence encoding a variantCry3Bb protein, 11098, similar in insecticidal activity to the variantCry3Bb protein 11231 in event MON853, but which is expressed moreefficiently from the modified coding sequence.

Corn seeds of the same hybrid species, with and without the respectivetransgenic events, were treated with either imidacloprid (available asGAUCHO® from Bayer Corp.) or tefluthrin (available as RAZE® fromWilbur-Ellis Co., Great Falls, Mont.; Walla Walla, Wash.) as follows. Aseed treatment formulation of the desired pesticide was prepared bymixing a measured amount in water as a carrier and applying theformulation for one minute at room temperature to a measured weight ofcorn seed in a rotostatic seed treater. The respective weights of thepesticide preparation and the corn seed were calculated to provide thedesired rate of treatment of pesticide on the seed. The pesticide wasmixed into sufficient water to permit efficient distribution of theformulation to all of the seeds in the batch while minimizing loss oftreatment formulation due to lack of uptake of the formulation by theseeds. Treated seeds were allowed to sit uncapped for at least fourhours before planting.

When the seeds were treated with imidacloprid, a sufficient amount ofGaucho® 600 FS (containing 48.7% by weight imidacloprid; available fromthe Gustafson LLC) was thoroughly mixed into water to form a seedtreatment formulation, and the formulation was applied to a weight ofcorn seed to provide treatment levels of 300 grams imidacloprid per 100kg of seed (.75 mg imidacloprid/kernel), or 400 grams imidacloprid per100 kg of seed (1.0 mg imidacloprid/kernel).

When the seeds were treated with tefluthrin, a sufficient amount ofRaze® 2.5 FS (containing 26.8% by weight tefluthrin; available fromWilbur-Ellis Co.,) was thoroughly mixed into water to form a seedtreatment formulation, and the formulation was applied to a weight ofcorn seed to provide treatment levels of 300 grams active tefluthrin per100 kg of seed (0.75 mg tefluthrin/kernel).

EXAMPLE 2

Field trials for the determination of efficacy of transgenic eventCry3Bb.11231 in corn seed in combination with corn root worm pesticideseed treatments against western and northern corn rootworm.

Field trials were run in accordance with pertinent protocols and inconformance with USDA notification requirements. The purpose of thetrials was to determine the efficacy of transgenic event Cry3Bb.11231 incorn seed in combination with corn root worm seed treatments againstwestern and northern corn root worm.

For each growing site that was selected, the plot design included thefollowing:

Row spacing: 30 inches Plot size: 4 rows × 20 Plant density: 2.0seed/foot Hybrid used: LH198 × LH185 or RX670 Replicates: 4 Design:Randomized complete block Locations: 4 Larvae source: naturalinfestations supplemented by artificial infestation of corn rootwormeggs at 400 eggs/ft (growth stage V2)

The following seed treatment combinations were used for each growingarea:

Pesticide and amount (grams AI/ No. Corn Seed Type 100 kg seed or mgai/kernel) 1 Isohybrid None, other than low levels for wire wormprotection 2 Cry3Bb.11231 None, other than low levels for wire wormprotection 3 Cry3Bb.11231 Gaucho ® 600 FS @ 300 gm AI/100 kg or .75 mgAI/kernel 4 Cry3Bb.11231 Gaucho ® 600 FS @ 400 gm AI/100 kg or 1.0 mgAI/kernel 5 Cry3Bb.11231 Raze ® 2.5 FS @ 300 gm AI/100 kg or .75 mgAI/kernel 6 Isohybrid Force ® 3G @ 0.014 gm AI/m, or 0.15 oz AI/1000 ftrow, applied as a 5″ band on the soil surface at the time of planting. 7Isohybrid Lorsban ® 15G (chlorpyrifos; available from DowElanco) @ 0.11gm AI/m, or 1.2 oz AI/1000 ft row, applied as a 5″ band on the soilsurface at the time of planting.

All seed treatments with pesticides were carried out as described inExample 1. In seed treatment number 1 and 2, Gaucho® was used for wireworm protection, but at levels sufficiently low that it would beexpected to have no effect on corn rootworms (i.e., at a treatment levelof about 60 gm of active/100 kg seed or 0.16 mg active/kernel),otherwise, seed receiving treatment number 2 had only transgenic eventCry3Bb.11231 and no pesticide treatment that would be expected to beeffective against corn rootworm.

For seeds having treatments numbered 3 through 5, the pesticides wereapplied by the methods described in Example 1. For seeds havingtreatment numbers 6 and 7, commercially available Force® 3G and Lorsban®15G were applied to the soil in a 5″ band at the time of sowing. Thelevels of application are as shown and are within the ranges recommendedfor standard commercial practice.

Corn seeds to be tested were planted and grown at four differentlocations across four Midwestern states in the United States corn beltaccording to the protocol described above.

The determination of damage by corn rootworm was made according to thefollowing protocol. At stage V4-V6, an evaluation of early stand wasmade by counting the number of plants per acre. At stage VT-R1, anevaluation of corn rootworm damage was carried out by methods that arewell known in the industry, and damage by corn rootworm was reportedaccording to the Iowa 1-6 rating system. In that system, the rootsystems of 10 corn plants per plot are recovered and scored using the1-6 rating scale, where: 1=no injury or only a few minor feeding scars,2=feeding injury evident, but no roots eaten back to 1½ inches of theplant, 3=at least one root eaten off to within 1½ inches of the plant,but never an entire node of roots destroyed, 4=one node of roots eatenback to within 1½ inches of the plant, 5=two nodes (circles) of rootseaten back to within 1½ inches of the plant, 6=three nodes (circles) ofroots eaten back to within 1½ inches of the plant.

TABLE 1 Corn rootworm damage to isohybrid corn plants havingconventional surface banding treatments and corn plants havingtransgenic event Cry3Bb.11231 alone and in combination with seedtreatment with selected pesticides at four growing locations. MEANS SEEDSITE SITE SITE SITE ACROSS NO. A B C D LOCATIONS 1 4.3 4.0 4.0 4.2 4.1 22.5 2.4 2.2 2.0 2.3 3 2.1 2.3 2.5 1.9 2.2 4 1.8 2.3 2.2 1.8 2.0 5 2.32.3 2.6 1.8 2.2 6 2.7 2.1 2.6 1.9 2.3 7 3.3 2.4 2.5 1.8 2.5

From the data of Table 1, it can be seen that transgenic seeds that weretreated with either imidacloprid or tefluthrin at any level were moreresistant to corn rootworm damage than the transgenic seeds without suchpesticide treatment. Moreover, all combination treatments (of transgenicevent plus pesticide treatment) were more efficacious that conventionalsurface banding with either FORCE® or LORSBAN®.

Therefore, it can be concluded that the treatment of a corn seed havinga transgenic event with either imidacloprid or tefluthrin providesimproved resistance over that provided by either the transgenic eventalone, or isohybrid seed that has also received a standard pesticidesurface banding treatment at planting.

EXAMPLE 3

Field trials for the determination of efficacy of transgenic eventCry3Bb.11231 in corn seed in combination with imidacloprid seedtreatments against western and northern corn rootworm.

A field trial was established and completed in accordance with pertinentprotocols and in conformance with USDA notification requirements. Thepurpose of the trial was to determine the efficacy of transgenic eventCry3Bb.11231 in corn seed in combination with corn rootworm seedtreatments using imidacloprid.

For each growing site that was selected, the plot design included thefollowing:

Row spacing: 30 inches Plot size: 4 rows × 20 Plant density: 2.0seed/foot Hybrid used: LH198 × LH185 or RX670 Replicates: 4 Design:Randomized complete block Locations: 4 Larvae source: naturalinfestations supplemented by artificial infestation of corn rootwormeggs at 400 eggs/ft (growth stage V2)

The following seed treatment combinations were used for each growingarea:

Pesticide and amount (grams AI/ No. Corn Seed Type 100 kg seed or mgai/kernel) 1 Isohybrid None, other than low levels for wire wormprotection 2 Cry3Bb.11231 None, other than low levels for wire wormprotection 3 Cry3Bb.11231 Gaucho ® 600 FS @ 300 gm AI/100 kg or .75 mgAI/kernel 4 Cry3Bb.11231 Gaucho ® 600 FS @ 400 gm AI/100 kg or 1.0 mgAI/kernel 5 Cry3Bb.11231 Raze ® 2.5 FS @ 300 gm AI/100 kg or .75 mgAI/kernel 6 Isohybrid Force ® 3G @ 0.014 gm AI/m, or 0.15 oz AI/1000 ftrow, applied as a 5″ band on the soil surface at the time of planting. 7Isohybrid Lorsban ® 15G (chlorpyrifos; available from DowElanco) @ 0.11gm AI/m, or 1.2 oz AI/1000 ft row, applied as a 5″ band on the soilsurface at the time of planting.

All seed treatments with pesticides were carried out as described inExample 1. In seed treatment number 1 and 2, Gaucho® was used for wireworm protection, but at levels sufficiently low that it would beexpected to have no effect on corn rootworms (i.e., at a treatment levelof about 60 gm of active/100 kg seed or 0.16 mg active/kernel),otherwise, seed receiving treatment number 2 had only transgenic eventCry3Bb.11231 and no pesticide treatment that would be expected to beeffective against corn rootworm.

For seeds having treatments numbered 3 through 5, the pesticides wereapplied by the methods described in Example 1. For seeds havingtreatment numbers 6 and 7, commercially available Force® 3G and Lorsban®15G were applied to the soil in a 5″ band at the time of sowing. Thelevels of application are as shown and are within the ranges recommendedfor standard commercial practice.

Corn seeds to be tested were planted and grown at four differentlocations across several Midwestern states in the United States cornbelt according to the protocol described above.

The determination of damage by corn root worm was made according to theprotocol described in Example 2.

TABLE 2 Corn rootworm damage to isohybrid corn plants and corn plantshaving transgenic event Cry3Bb.11231 alone and in combination with seedtreatment with imidacloprid pesticide at different growing locations.CORN ROOTWORM DAMAGE IN EACH PERCENT IOWA CLASS (IOWA 1-6 SCALE) GRANDOF TREATMENT 1 2 3 4 5 6 TOTAL CONTROL Isohybrid 0 3 16 36 21 4 80 100Cry3Bb.11231 5 51 23 1 0 0 80 31.2 Imidacloprid @ 400 g/100 kg of seed 315 36 21 5 0 80 80.5 Cry3Bb.11231 with 13 53 14 0 0 0 80 18.2Imidacloprid @ 400 gm/100 kg of seed FORCE ® 3G surface band at planting3 58 34 3 0 0 98 39.2 LORSBAN ® 15G surface band at planting 6 39 38 161 0 100 57.1 Notes: a. Data for the isohybrid control was taken as thesame as determined for a related protocol that was carried out in anadjoining plot.

The data showed that both the transgenic event alone and seed treatmentwith imidacloprid alone provided some level of protection against cornrootworm damage above the untreated isohybrid control. At higher levelsof damage (i.e., damage levels 4-6), corn having the transgenic eventsuffered 4.7% of the damage of the non-transgenic control. Since 4.7%would be considered to be about 5%, the Cry3Bb.11231 event wasconsidered to be within a preferred effectiveness range of about 5% toabout 50% of the damage of the non-transgenic control.

Imidacloprid seed treatment alone at 400 gm/100 kg was effective againstcorn rootworm damage, but the effectiveness of imidacloprid was lowerthan the effectiveness of the transgenic event alone. The combination oftreatment with imidacloprid of the transgenic seed was more effectiveagainst rootworm damage than the pesticide treatment alone or thetransgenic event alone. Moreover, the combination of Cry3Bb.11231 withimidacloprid at 400 gm/100 kg of seed provided better protection thanthe commercial standard treatment of either FORCE® or LORSBAN® appliedas a surface band at planting.

The advantages of the present treatment of transgenic seed withimidacloprid include the simplification of planting, by removing therequirement for separate application of the pesticide. Furthermore,planting is easier and safer, since the planter does not have to handlea concentrated pesticide.

The combination of imidacloprid seed treatment with corn seed having a

Cry3Bb.11231 transgenic event was tested for possible synergy at a levelof rootworm damage of 3-6. In the first test, shown in Table 2, thepercentage of test plants having damage levels of from 3 to 6, on theIowa 1-6 Scale, was determined for the control and for seeds treatedwith the pesticide at two levels, and for seeds having the transgenicevent, alone and in combination. The following formula was then used tocalculate a “synergy threshold”:(% of control Cry3Bb.11231)*(% of control imidacloprid treatment)/100.

This threshold was compared against the percent of control for thetreatment combination (i.e., Cry3Bb.11231 with imidacloprid @ 400 gm/100kg). If the treatment combination percent of control was below thethreshold, then it was concluded that there was synergy. If thetreatment combination percent of control was above the threshold, thenit was concluded that synergy was not demonstrated for that combination.

It was believed that the measurement of rootworm damage at higher damagelevels (i.e., levels 3-6) is a useful indicator that correlates withsubsequent yield loss due to such damage. The reason for this is thatrootworm damage at levels 1 and 2 seldom causes corn plants to fall overand lodge, and such minimal root loss is not believed to reduce thenumber or weight of kernels per ear. However, root damage at levels of 3and above increasingly causes lodging and loss of yield. Therefore, itis believed that the summed damage levels of 3-6 (and in some cases, 4-6and 5 and 6), provides a useful indication of the effect of cornrootworm damage on subsequent corn yield.

TABLE 3 Efficacy of seed treatment with imidacloprid alone and incombination with corn transgenic event Cry3Bb.11231 against cornrootworm damage at levels 3-6 on the Iowa 1-6 Scale. NUMBER OF PERCENTTHRESH- PLANTS HAVING OF OLD TREATMENT 3-6 DAMAGE LEVEL CONTROL SYNERGYUntreated 96.1 100 — Control Cry3Bb.11231 40 31.2 — Imidacloprid @ 71.780.5 — 400 gm/100 kg Cry3Bb.11231 24 18.2 25.1 with imidacloprid @ 400gm/100 kg FORCE 3G 40.7 39.2 — as surface band LORSBAN 15G 60.8 57.1 —as surface band

This analysis indicated that the combination of the corn Cry3Bb.11231transgenic event with seed treatment with imidacloprid at 400 gm/100 kgwas synergistic and unexpectedly efficacious against corn rootwormdamage at the 3-6 level. Accordingly, it was concluded that thecombination of the transgenic event with the imidacloprid seed treatmentprovided significant advantages over the use of either method alone, andthat such protection was unexpectedly superior in efficacy againstsevere damage by corn rootworm.

It was also believed to be noteworthy that the combination ofimidacloprid and transgenic event provided protection against severecorn rootworm damage at levels that were far better than that providedby either FORCE® or LORSBAN® applied as surface bands.

EXAMPLE 4

Field trials for the determination of efficacy of transgenic eventCry3Bb.11231 in corn seed in combination with tefluthrin pesticide seedtreatments against western and northern corn rootworm.

A field trial for the determination of efficacy of the combination oftransgenic event Cry3Bb.11231 in corn seed with tefluthrin (available asRAZE® from Wilbur-Ellis Company) could be carried out according to thesame protocol as described in Example 3, except that tefluthrin would besubstituted for imidacloprid in each case where imidacloprid was used atlevels expected to be effective against corn rootworm (e.g., at levelsof higher than 60 gm/100 kg seed). If desirable, it would be permissibleto continue to use imidacloprid at levels of 60 gm/100 kg, or less, forwireworm protection.

It would be expected that the combination of tefluthrin seed treatmentwith a transgenic event in corn seed having effectiveness against cornrootworm would provide synergistic protection similar to that shown inExample 3 for the combination of imidacloprid and Cry3Bb.11231.

EXAMPLE 5

This example illustrates the use of a seed mixture containing variousratios of transgenic and non-transgenic seeds to deploy a transgenicrefuge, with or without seed treatments, provides an effective means forallowing adequate survival of susceptible corn rootworms in fields ofrecombinant crops to prevent or slow the rate of resistance evolutionand still reduce economic loss due to corn rootworm infestation.

The western corn rootworm (WCR), D. virgifera virgifera, is a widelydistributed pest of maize in North America. In many instances,insecticides are indiscriminately used to reduce their numbers below aneconomically damaging level. To assist in the reduction of insecticidesused against the WCR, the inventors herein have utilized a transgenicline of maize expressing the Cry3Bb insecticidal protein. Upon ingestionby the rootworm, this protein forms pores in the midgut cells causingswelling and lysis of these cells and eventually death to the feedingworm. One concern is that the WCR will evolve resistance to the proteinwhich will potentially spread throughout the rootworm's distribution andpopulation. Deploying a transgenic refuge by planting seed mixtures oftransgenic and non-transgenic maize may be a reliable resistancemanagement strategy for controlling corn rootworms. The inventors hereinhave investigated the technical feasibility of a resistance managementprogram that uses in-field seed mixes containing various proportions oftransgenic and non-transgenic seed, i.e., T:NT, in combination with anew seed treatment technology to prevent substantial damage tonon-transgenic maize provided in the mix. If effective, this methodologycould provide growers with greater yields at lower cost and laborrequirements, and could simultaneously provide a means for preventing ormanaging the development of resistant strains of CRW. The underlyingassumption is that planting of a mix comprising transgenic vs.non-transgenic (T:NT) seed at the appropriate ratios allows adequatesurvival of susceptible CRW in the fields to prevent or slow the rate ofresistance evolution and still reduce economic losses due to CRWinfestation.

This method utilized a factorial design having five ration levels oftransgenic vs non-transgenic seed in a mix, consisting of 100:0 T:NT,90:10 T:NT, 80:20 T:NT, 60:40 T:NT, and 0:100 T:NT. Two levels of WCRegg infestation were utilized at the V2-V3 plant growth stage,consisting of 500 eggs per thirty centimeter row and 1000 eggs perthirty centimeter row, which were designated as low and high infestationrates, respectively. Two levels of seed treatment were utilized, similarto what was used in the examples above. One treatment level consisted ofGaucho (imidocloprid) at 60 grams per 100 kilogram of seed and wasdesignated as WWST. The other treatment level consisted of clothianidineat 200 milligrams on non-transgenic (NT) seed, Gaucho on the transgenicseed (T), designated CRWST1, and 100% non-transgenic (NT) seed mix.

Four additional treatments were used for comparison purposes only, andwere not included at all in the ANOVA's. One of these additionaltreatments consisted of T80NT20 at a low and high level of egginfestation, with imidocloprid applied at 30 grams per 100 kilograms ofseed on transgenic (T) seed and Gaucho on the non-transgenic seed (NT),designated as the CRWST2 treatments. Another of these additionaltreatments consisted of two 100% non-transgenic (NT) trials at low andhigh levels of egg infestation, treated only with Force3G insecticide,which is the conventional means presently in commercial use for treatingcorn rootworm infestation. All treatments were replicated four timesover 96 plots, and the seeds were hand planted to verify the propertransgenic vs non-transgenic (T:NT) rations. Emergence cages coveredfive plants, exemplifying the total plot of T:NT at various ratios. AHills & peters 1-6 damage rating scale, as indicated herein, was used toscore the damage to roots near the end of the adult emergence cycle,using ten plants per rep out of a total of 800 plants.

Over all of the treatments, significantly more female WCR emerged thanmale WCR (4972 female vs 2823 male), using a paired t-test, in whicht=−7.82, df=79, and P<0.0001. It was determined that there was nosignificant interactions among seed treatments, egg rates, and ratios oftransgenic to non-transgenic maize. Seed treatment had no significanteffect on the mean number of WCR emerging, however, it was determinedthat significantly more (F=18.65, df=1.57, P<0.0001) WCR emerged fromcaged infested at the high level (4447 total, 111.2±19.4) than at thelow level of infestation (3348 total, 83.7±18.3). The mean number of WCRemerging from the different seed ratios differed significantly(F=105.34, df=4.57, P<0.01). All pairwise comparisons were significantlydifferent (df=57, P<0.0001) based on t-tests on Lease Squares Meansusing Bonferroni adjustments to control Type 1 errors (alpha=0.05),except for WCR emerging from the T90:NT10 and T80:NT20 ratio studies.The fewest number of WCR emerged from the T100NT0 ratio study and thehighest number from the T0NT100 ratio study. The mean number of WCRemerging from the T80NT20 maize treated blend with CRWST2 (29.3±6.2) wascomparable to the mean number emerging from the T80NT20 maize (35.9±4.8)treated blend with CRWST1 and WWST. The mean number of WCR emerging fromthe maize treated with Force3G (98.3±16.6) was comparable to the meannumber emerging from the T60NT40 ratio study (93.6±12.2).

Seed treatment had no significant effect on mean root damage rating,however, it was determined that a significant interaction between egginfestation rates and ratios of transgenic to non-transgenic maize(F=5.35, df=1.776, P<0.001). Based on t-tests on Least Squares Meansusing Bonferroni adjustments to control Type 1 errors (alpha=0.05), mostpairwise comparisons were significantly different (df=776, P<0.0001).Exceptions include root damage rating from the low egg infestation rateat T0:NT100 and high egg infestation rate at T0:NT100, low egginfestation rate at T100:NT0, and high egg infestation rate at T100:NT0.The lowest root damage ratings were obtained from the T100NT0 ratiostudies and the highest root damage rating was observed in the T0NT100ratio study. The mean root damage rating from the maize treated withCRWST2 (1.61±0.10) was comparable to the mean root damage rating fromthe T80NT20 ratio study (1.81±0.07). Similarly, the mean root damagerating from the maize treated with Force3G (2.81±0.09) was comparable tothe mean root damage rating from the T60NT40 ratio study (2.74±0.09).

More females emerged than males. Whether this is due to differentialmortality on the sexes caused by the transgenic maize or some otherphenomenon is not clear.

Further investigations into the sex ratio of WCR is necessary toelucidate any sexually biased effects caused by the transgenic maize.

The number of emerging WCR differed among the ratios of transgenic maizeto non-transgenic maize. The ratios T100NT0, T90NT10, and T80NT20 werethe most effective at reducing rootworm populations. These three ratioshad the least number of emerged beetles. As expected, the non-transgenicmaize had little or no controlling effect on beetle numbers. The CRWST1had no significant impact on reducing the number of emerging WCR or onroot damage rating. Similar numbers of WCR emerged from both the T60NT40ratio studies and the maize treated with Force3G, which may explain thesimilar amount of damage to maize roots for these two treatments.

Root damage greatly exceeded economically acceptable levels (RDR 3.0)for the T0NT100 maize plots, and only slightly for the T60NT40 ratiostudies at high egg infestation rates. The least amounts of root damageoccurred to plants in the T100NT0, T90NT10, and T80NT20 ratio studies.Maize planted at these ratios never exceeded the economically damagingroot damage rating level of 3.0 on the Iowa Hills&Peters scale.

One concern about the commercial release of transgenic maize for controlof CRW is the evolution of resistance by the rootworms. One means formanaging the development of resistance is to require that producers andgrowers plant a refuge to maintain resistant alleles at a low frequency.This disclosure illustrates a seed mix refuge option. The data in thisexample illustrates that a T90NT10 and a T80NT20 ratio seed mixmaintained root damage levels below the economically damaging levels andproduced similar numbers of adult beetles. A T60NT40 ratio only exceededeconomically damaging levels under high levels of insect infestation andwas comparable to the conventionally used insecticide Force3G. Thecombination of a seed treatment along with the deployment of refuge seedin a mix of transgenic seeds is therefore a useful strategy forprolonging the onset of resistance to either the seed treatment or tothe recombinant insect inhibitory trait contained within the planttissue.

These results demonstrate that all ratios including transgenic maizewere as effective as the traditional method of applying insecticides tomaintain WCR root damage levels below economically damaging levels. Mostof the transgenic:non-transgenic ratios performed much better than thetraditional method. Only the 100% non-transgenic maize had consistentroot damage ratings exceeding the economic threshold. Using a seed mixof transgenic and non-transgenic seed in various proportions, inparticular in combination with seed treatments providing a second modeof action, for planting in a crop in a field, can reduce the onset ofresistance in the target insect pests.

EXAMPLE 6

This example illustrates a few of the various combinations of types offirst recombinant or first transgenic crop seed that can be present in aseed blend with various types of transgenic or non-transgenic refugecrop seed or other refuge seed. The skilled artisan will recognize themany various combinations available in the art based on the presentdisclosure.

A transgenic insect resistant crop seed that contains a transgeneconferring herbicide tolerance to the plant grown from the transgeniccrop seed is mixed in various proportions with a refuge seed. The refugeseed is also a transgenic seed but contains only a transgene conferringherbicide tolerance to a plant grown from the seed, such as resistanceto glyphosate, resistance to basta, resistance to glufosinate, and thelike, and is not transgenic with respect to any insect resistance trait.However, the refuge seed is not required to contain a transgene. Forexample a transgenic cotton crop seed containing a transgene encoding aBt insecticidal resistance gene toxic to lepidopteran insect larvae ispresent in a seed blend with a refuge cotton seed that contains arecombinant gene that confers resistance to various concentrations ofROUNDUP herbicide when applied to the surface of the plants. Thetransgenic cotton crop seed containing the Bt insecticidal resistancegene also contains a recombinant gene that confers resistance to variousconcentrations of ROUNDUP herbicide. Both types of seeds may be coatedwith a seed treatment that contains a pesticidal agent that is differentfrom the Bt insecticide present in the transgenic cotton crop seeds. Theseeds are blended together and bagged and provided to a grower. Thegrower plants the seeds in a field, and the seeds sprout and developinto plants. Plants are sprayed with an appropriate herbicide to controlvolunteer growth and development and/or weed pest growth and infestationin the field. The herbicide tolerant insect sensitive plants grown fromthe refuge seed act as a refuge for cotton insect pest infestation, andare evenly dispersed throughout the field.

A first transgenic maize variety is transformed to contain a geneconferring resistance to a coleopteran insect, a gene conferringresistance to a lepidopteran insect, a gene conferring tolerance ROUNDUPherbicide, and an gene causing the lipid composition of the oil in thecorn seed crop to be different than the oil composition in a nativemaize variety. Seeds of the first transgenic maize variety are blendedtogether in a uniform mixture with refuge seeds. Refuge seeds may be amixture of transgenic and non-transgenic seeds or a uniform compositionof either transgenic or non-transgenic seeds. When refuge transgenicseeds are used, a mixture of refuge transgenic maize seed containing atleast one gene conferring tolerance to the herbicide ROUNDUP and eitherno other transgene or only one other transgene selected from the groupconsisting of the same gene conferring resistance to a coleopteraninsect that is present in the first transgenic maize variety and thesame gene conferring resistance to a lepidopteran insect that is presentin the first transgenic maize variety. All of the seeds comprising therefuge seeds have been coated with a seed treatment that contains aninsecticide that confers at least a second mode of action different fromeither the transgene that confers coleopteran insect resistance or thetransgene that confers lepidopteran insect resistance to the firsttransgenic maize variety. The first transgenic maize seed and the refugeseed are blended together and bagged and provided to a grower. Thegrower plants the seeds in a field, and the seeds sprout and developinto plants. Plants are sprayed with an appropriate herbicide to controlvolunteer growth and development and/or weed pest growth and infestationin the field. The herbicide tolerant, insect infestation sensitiveplants grown from the refuge seed act as a refuge for maize insect pestinfestation, and are evenly dispersed throughout the field.

All references cited in this specification, including without limitationall papers, publications, presentations, texts, reports, manuscripts,brochures, internet postings, journal articles, periodicals, and thelike, are hereby incorporated by reference. The discussion of thereferences herein is intended merely to summarize the assertions made bytheir authors and no admission is made that any reference constitutesprior art. Applicants reserve the right to challenge the accuracy andpertinence of the cited references.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

What is claimed is:
 1. A method of managing pest resistance in a plot ofpest resistant crops, said method comprising: a) providing seed of afirst transgenic crop, the first transgenic crop expressing a firsttransgene and a second transgene, the first transgene providingincreased tolerance or resistance to at least one Coleopteran pest andthe second transgene providing resistance to at least one Lepidopteranpest; b) providing seed of a second transgenic crop, the secondtransgenic crop expressing a third transgene, the third transgeneproviding resistance to at least one Coleopteran pest, wherein the thirdtransgene is the same transgene as the first transgene and wherein thesecond transgenic crop does not comprise a transgene having pesticidalactivity against a Lepidopteran pest; and c) providing the seed of thefirst transgenic crop and the seed of the second transgenic crop forplanting, wherein the seed is planted in the plot; wherein said firsttransgenic crop and said second transgenic crop comprise a herbicidetolerance transgene conferring tolerance to glyphosate; and wherein pestresistance is managed relative to a plot lacking said second transgeniccrop.
 2. The method of claim 1, wherein the at least one Coleopteranpest is a corn rootworm pest.
 3. The method of claim 1, wherein the atleast one Lepidopteran pest is selected from the group consisting ofcorn borers, earworms, armyworms and cutworms.
 4. The method of claim 1,further comprising treating said first transgenic crop seed and/or saidsecond transgenic crop seed with a pesticidal agent.
 5. The method ofclaim 1, wherein the first transgene, second transgene and thirdtransgene encode a Bacillus thuringiensis Cry protein.